SOCS-1 gene methylation in cancer

ABSTRACT

Methods are provided for identifying a cell exhibiting unregulated growth associated with methylation-silenced transcription of a suppressor of cytokine signaling (SOCS)/cytokine-inducible SH2 protein (CIS) family member (SOCS/CIS) gene such as the SOCS-1 gene. In addition, methods of treating a cancer patient, wherein cancer cells in the patient exhibit methylation-silenced transcription of SOCS/CIS gene such as a SOCS-1 gene, are provided, as are reagents for practicing such methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 10/123,882 filed Apr. 15, 2002, now U.S. Pat. No. 7,348,139; whichclaims the benefit under 35 USC §119(e) to U.S. Application Ser. No.60/283,709 filed Apr. 13, 2001, now abandoned. The disclosure of each ofthe prior applications is considered part of and is incorporated byreference in the disclosure of this application.

GRANT INFORMATION

This invention was made with government support under Grant No. CA 58184awarded by the National Cancer Institute. The government has certainrights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to an epigenetic markerindicative of cancer, and more specifically to the identification of acorrelation of methylation-silenced transcription of SOCS-1 geneexpression and cancer, and to reagents and methods for detecting andtreating a cancer associated with methylation-silenced transcription ofSOCS-1 gene expression.

2. Background Information

Cancer can occur due to contact with various etiologic agent, including,for example, exposure to environmental carcinogens or infection by avirus, or can be acquired congenitally from one or both parents. Suchcancers have a genetic basis in that the changes responsible for thecancer are at the level of the nucleotide sequence of one or more genesin an individual. For example, some viruses have a life cycle thatincludes a stage in which it integrate into the nuclear genome of anorganism such as a human. Where such integration results in disruptionof a gene that is involved, for example, in instructing a cell to stopproliferating, the result can be unregulated proliferation of the cell,as is characteristic of some cancer cells.

In comparison, chemical carcinogens can cause physical damage to the DNAin an individual. Where the damage caused by the carcinogen is minimal,DNA repair mechanisms often can repair the damage such that the repairedsequence is identical to the sequence prior to the damage. Where thedamage caused by the carcinogen is extensive, the cell containing thedamaged DNA often dies. In some cases, however, the damage is notsufficient to kill the cell, but is too extensive to be repairedproperly. In such cases, while the effort by the cell to repair thedamaged DNA can be sufficient for the cell to continue growing anddividing, the repaired sequence is different from the DNA sequence priorto the damage. Where the defective repair occurs in a gene, the productof that gene may not be produced or, if the gene product is produced, itmay not function properly. As such, where the normal gene product may,for example, regulate the time the cell normally would be destined todie, a cell lacking the normal gene product, as well as progeny of thecell, may continue to proliferate in an unregulated manner, as ischaracteristic of some cancer cells.

In addition to such genetic changes, cancer also can be caused byepigenetic mechanisms, which do not result in mutations of the DNAsequence. The most commonly observed epigenetic mechanism involvessilencing of gene expression due to methylation of the gene sequence.Methylation of cytosine residues located 5′ to guanosine in CpGdinucleotides, particularly in CpG-rich regions (CpG islands), often isinvolved in the normal regulation of gene expression in highereukaryotes. For example, extensive methylation of CpG islands isassociated with transcriptional inactivation of selected imprintedgenes, as well as the genes on the inactivated X chromosome in females.Aberrant methylation of normally unmethylated CpG islands also has beenfound in immortalized and transformed cells, and has been associatedwith transcriptional inactivation of defined tumor suppressor genes inhuman cancers.

Changes to genes that are associated with cancer, including mutationsthat result in loss of expression of gene or expression of a defectivegene product, and epigenetic mechanisms such as methylation-silencing ofgene transcription, provide markers useful for determining whether acell is susceptible to loss of normal growth control and, therefore,potentially a cancer cell. For example, a mutation of the BRCA1 gene hasbeen associated with breast cancer. As such, diagnostic tests can beperformed using cells, for example, from a woman with a family historyof breast cancer to determine whether the woman has the BRCA1 mutationthat is a marker for breast cancer. The prostate specific antigen (PSA)is another example of a marker, in this case for prostate cancer.Although neither the defect resulting in expression of the PSA nor thenormal function of PSA in the body is known, PSA nevertheless provides avaluable cancer marker because it allows the identification of menpredisposed to prostate cancer or at a very early stage of the diseasesuch that effective therapy can be implemented.

Cancer often is a silent disease that does not present clinical signs orsymptoms until the disease is well advance. As such, the use of markersthat allow the identification of individuals susceptible to a cancer, oreven that allow detection of a cancer at an early stage, can be of greatbenefit. Unfortunately, such markers are not available for most cancers.As such, many cancer patients do not seek medical assistance until thecancer is at a stage that requires radical therapy, or is untreatable.Thus, a need exists for markers that can be used to detect cancer cells.The present invention satisfies this need and provides additionaladvantages.

SUMMARY OF THE INVENTION

The present invention is based on the identification of a correlation ofunregulated cell growth as occurs in various cancers, andmethylation-silenced transcription of a suppressor of cytokine signaling(SOCS)/cytokine-inducible SH2 protein (CIS) family member (SOCS/CIS)gene. This correlation is exemplified by the demonstration thatmethylation-silenced transcription of the SOCS-1 gene is found invarious cancers, including hepatocellular carcinoma, multiple myeloma,and acute leukemias, and that methods and reagents that restore SOCS-1gene expression is such cancer cells result in apoptosis of the cancercell. As such, the present invention provides methods of diagnosing acancer by detecting methylation-silenced transcription of a SOCS/CISgene, as well as methods of treating such cancers.

The present invention relates to a method for identifying a cell thatexhibits, or is predisposed to exhibiting, unregulated growth. Such amethod can be performed by detecting methylation of a cytosine residuein a CpG dinucleotide in a CpG island of a SOCS/CIS gene in a test cell,or an extract comprising nucleic acid molecules of the test cell,wherein the SOCS/CIS gene methylation results in a reduced level oftranscription of the gene in the test cell as compared to acorresponding normal cell, i.e., a corresponding cell that exhibitsnormal regulated growth. The SOCS/CIS gene can be a gene encoding anymember of the SOCS/CIS family of protein, including, for example, a SOCSgene such as a SOCS-1, SOCS-2 (also called STATI2) or SOCS-3 gene, or acytokine-inducible SH2 protein-2 (CIS2) gene.

The cell exhibiting, or predisposed to exhibiting, unregulated growthgenerally, but not necessarily, is a neoplastic cell, which can be apremalignant cell or a malignant cell, i.e., a cancer cell, for example,a hepatocellular carcinoma cell, a multiple myeloma cell, or an acuteleukemia cell. The test cell can be any cell, including, for example, aprimary cell that has been obtained from a subject and is placed inculture or is being adapted to grow in culture, wherein a method of theinvention provides a means to determine whether the cell has maintainedregulated growth control and, therefore, following expansion, issuitable for readministration to the subject. The test cell also can bea cell that is obtained from a subject, for example, a cell obtainedfrom an organ sample, a tissue sample, or a cell sample, which can beobtained by a biopsy procedure. As such, the test cell can be a cellfrom a liver sample, a skin sample, a lymph node sample, a kidneysample, a lung sample, a muscle sample, a bone sample, or a brainsample. The cell also can be a component of a biological fluid, forexample, bone marrow, blood, lymph, cerebrospinal fluid, saliva, sputum,stool, urine, or ejaculate. A method of the invention also can bepracticed using an extract of a test cell, wherein the extract includesnucleic acid molecules of the test cell, particularly genomic DNA.

Methylation of a CpG dinucleotide in a CpG island of a SOCS/CIS gene canbe detected using any of various well known methods for detecting CpGmethylation of a nucleic acid molecule. For example, such methylationcan be detected by contacting a nucleic acid molecule, which includesall or a portion of a CpG island of the SOCS/CIS gene sequence, with amethylation sensitive restriction endonuclease. The methylationsensitive restriction endonuclease can be one that cleaves a recognitionsite containing a methylated cytosine residue of a CpG dinucleotide inthe SOCS/CIS gene sequence, for example, a restriction endonuclease suchas Acc III, Ban I, BstN I, Msp I, or Xma I, whereby cleavage of thenucleic acid molecule indicates that the SOCS/CIS gene in the test cellis methylated. Conversely, the methylation sensitive restrictionendonuclease can be one that cleaves a recognition site containing a CpGdinucleotide in the SOCS/CIS gene sequence only when the cytosineresidue of the CpG dinucleotide is unmethylated, for example, arestriction endonuclease such as Acc II, Ava I, BssH II, BstU I, Hpa II,or Not I, whereby a lack of cleavage of the nucleic acid moleculeindicates that SOCS/CIS gene in the test cell is methylated.

Methylation of a CpG dinucleotide in a CpG island of a SOCS/CIS genealso can be detected by contacting a nucleic acid molecule comprisingthe SOCS/CIS gene of the test cell with a chemical reagent thatselectively modifies either an unmethylated cytosine residue or amethylated cytosine residue, and detecting a product generated due tocontact with the reagent, wherein the product is indicative of themethylation status of a cytosine residue in a CpG dinucleotide of theSOCS/CIS gene sequence, i.e., whether the cytosine residue is methylatedor unmethylated. Depending on the particular chemical reagent used insuch a method and, therefore, the effect that the reagent has on aSOCS/CIS gene sequence containing a methylated or unmethylated cytosineresidue in a CpG dinucleotide, the product generated according to themethod, which generally is a nucleic acid product that can, but neednot, contain a modified nucleotide, can be detected, for example, bysequencing the product, or by a method such as electrophoresis,chromatography, mass spectrometry, or a combination thereof.

In one embodiment, the chemical reagent used in a method of theinvention is hydrazine, thereby producing a hydrazine treated SOCS/CISgene sequence. Where the chemical reagent is hydrazine, the method ofdetecting CpG dinucleotide methylation of a SOCS/CIS gene sequencefurther includes contacting the hydrazine treated SOCS/CIS gene sequencewith a reagent such as piperidine, which cleaves the nucleic acidmolecule at hydrazine modified cytosine residues to generate a productcomprising fragments of the SOCS/CIS gene sequence; separating thefragments according to molecular weight, and detecting a gap in thefragment separation pattern at a position known to contain a cytosineresidue in a SOCS/CIS gene sequence, wherein the gap is indicative ofmethylation of a cytosine residue in the CpG dinucleotide in theSOCS/CIS gene of the test cell.

In another embodiment, the chemical reagent used in a method of theinvention is a composition comprising bisulfite ions, for example,sodium bisulfite, whereby unmethylated cytosine residues in the SOCS/CISgene sequence are converted to bisulfite modified cytosine residues.Where the chemical reagent comprises bisulfite ions, the method ofdetecting CpG dinucleotide methylation of a SOCS/CIS gene sequencefurther includes exposing the bisulfite ion treated SOCS/CIS genesequence to alkaline conditions, whereby bisulfite modified cytosineresidues are converted to uracil residues; and detecting the amount ordistribution of uracil residues in the bisulfite ion treated SOCS/CISgene of the test cell, wherein a decrease in the amount or distributionof uracil residues in the SOCS/CIS gene from the test cell, as comparedto the amount or distribution of uracil residues in a correspondingbisulfite ion treated unmethylated SOCS/CIS gene following exposure toalkaline conditions, is indicative of methylation of cytosine residuesin CpG dinucleotides in the SOCS/CIS gene of the test cell.

The amount or distribution of uracil residues produced according to sucha method can be detected using any of various methods. For example, theamount or distribution of uracil residues can be detected by sequencingthe bisulfite modified SOCS/CIS gene sequence following exposure toalkaline conditions, and identifying uracil residues in the sequence.The amount or distribution of uracil residues also can be detected bycontacting the bisulfite ion treated SOCS/CIS gene sequence, followingexposure to alkaline conditions, with an oligonucleotide thatselectively hybridizes to a SOCS/CIS gene sequence containing uracilresidues, and detecting selective hybridization of the oligonucleotide.Selective hybridization of the oligonucleotide can be detected, forexample, by performing the method using an oligonucleotide that includesa detectable label (e.g., a radioisotope, a paramagnetic isotope, aluminescent compound, a chemiluminescent compound, a fluorescentcompound, a metal chelate, an enzyme, a substrate for an enzyme, areceptor, or a ligand for a receptor) and detecting the label in ahybridization product or a derivative thereof. Selective hybridizationalso can be detected, for example, by utilizing the oligonucleotide as asubstrate for a primer extension reaction, and detecting a product ofthe primer extension reaction.

The amount or distribution of uracil residues in a bisulfite ion treatedSOCS/CIS gene sequence following exposure to alkaline conditions alsocan be detected using an amplification reaction, for example, apolymerase chain reaction (PCR). In one embodiment, the amplification isperformed by contacting the SOCS/CIS gene sequence with an amplificationprimer pair comprising a forward primer and a reverse primer underconditions suitable for amplification, wherein at least one primer ofthe primer pair comprises an oligonucleotide that selectively hybridizesto a SOCS/CIS gene sequence containing uracil residues, wherebygeneration of an amplification product is indicative of methylation ofcytosine residues in CpG dinucleotides in the SOCS/CIS gene of the testcell. In another embodiment, the amplification reaction is performed bycontacting the SOCS/CIS gene sequence with an amplification primer paircomprising a forward primer and a reverse primer under conditionssuitable for amplification, wherein both primers of the primer pairselectively hybridize to a SOCS/CIS gene sequence containing cytosineresidues, but not to a SOCS/CIS gene sequence containing uracilresidues, whereby generation of an amplification product is indicativeof a lack of methylation of cytosine residues in CpG dinucleotides inthe SOCS/CIS gene of the test cell.

In still another embodiment, the amplification reaction for detectingthe amount or distribution of uracil residues in a bisulfite ion treatedSOCS/CIS gene following alkaline treatment is performed by contactingthe SOCS/CIS gene sequence with a first amplification primer pair and asecond amplification primer pair under conditions suitable foramplification, wherein the first amplification primer pair is amethylation-specific primer pair comprising a forward primer and areverse primer, wherein at least one primer of the first primer paircomprises an oligonucleotide that selectively hybridizes to a SOCS/CISgene sequence containing uracil residues, and wherein the secondamplification primer pair is an unmethylation-specific primer paircomprising a forward primer and a reverse primer, wherein both primersof the second primer pair selectively hybridize to a SOCS/CIS genesequence containing cytosine residues, but not to a SOCS/CIS genesequence containing uracil residues, and wherein an amplificationproduct, if any, generated by the first primer pair has a first length,and an amplification product, if any, generated by the second primerpair has a second length, which is different from the first length,whereby the length of the amplification products is indicative of theamount or distribution of uracil residues and, therefore, of methylationof cytosine residues in CpG dinucleotides in the SOCS/CIS gene of thetest cell.

The methods of the invention are particularly adaptable to beingperformed in a high throughput format, wherein a plurality of testcells, or extracts of the test cells, or test cells and extracts thereofare examined sequentially, in parallel, or a combination thereof. Eachof the test cells, or extracts of the test cells, of a plurality beingexamined can the same or different, or can be a combination thereof. Forexample, the method can be practiced using duplicate or triplicatesamples of each of two or more different test cells, i.e., test cellsobtained from different subjects or from different sites of a singlesubject, for example, from a site of a cancer, or a site adjacent to acancer, or a surgical margin remaining after removal of a cancer; and,if desired, can further include cells that correspond to the test cells,but exhibit normally regulated growth, such cells providing controls orstandards with which to compare results obtained for a test cell.Generally, the plurality of test cells, or extracts of the test cells,are arranged in an array, particularly an addressable array, thusproviding a means to correlate a result with the source of the testcells. An array can be produced on a microchip, a glass slide, a bead,or other such solid support that allows each sample in the array to besubstantially isolated from each other sample, and conveniently can beused in an automated system for adding or removing reagents forpracticing the method or for detecting a result of the method.

The present invention also relates to method for identifying aneoplastic cell, which exhibits unregulated growth. Such a method can beperformed by detecting methylation of a cytosine residue of a CpGdinucleotide in a CpG island of a suppressor of cytokine signaling-1(SOCS-1) gene in a sample comprising a test cell, or an extract thereof,whereby methylation of the SOCS-1 gene results in a reduced level oftranscription and, therefore, expression of the SOCS-1 gene product inthe test cell as compared to a corresponding normal cell.

The neoplastic cell can be a premalignant cell or a malignant cell,i.e., a cancer cell, for example, a hepatocellular carcinoma cell, amultiple myeloma cell, or an acute leukemia cell. The sample to beexamined according to a method of the invention can be a sample obtainedfrom a subject such as a human subject, a domesticated animal, a farmanimal, or the like, and can be any sample containing a cell suspectedof being a neoplastic cell, or containing nucleic acid moleculesincluding a SOCS-1 gene sequence or a portion thereof comprising a CpGisland. As such, the sample can be an organ sample, a tissue sample, ora cell sample, for example, a liver sample, a skin sample, a lymph nodesample, a kidney sample, a lung sample, a muscle sample, a bone sample,or a brain sample; or can be a sample of a biological fluid, forexample, bone marrow, blood, serum, lymph, cerebrospinal fluid, saliva,sputum, stool, urine, or ejaculate.

Methylation of a CpG dinucleotide in a SOCS-1 gene can be detected bycontacting a nucleic acid molecule comprising the SOCS-1 gene of thetest cell with a methylation sensitive restriction endonuclease thatcleaves a recognition site comprising a methylated cytosine residuecomprising a CpG dinucleotide, whereby cleavage of the nucleic acidmolecule is indicative of methylation of the SOCS-1 gene in the testcell, or with a methylation sensitive restriction endonuclease that doesnot cleave a recognition site comprising a methylated cytosine residuecomprising a CpG dinucleotide, whereby a lack of cleavage of the nucleicacid molecule is indicative of methylation of the SOCS-1 gene in thetest cell.

Methylation of a CpG dinucleotide in a SOCS-1 gene also can be detectedby contacting a nucleic acid molecule comprising the SOCS-1 gene of thetest cell with a chemical reagent that selectively modifies either anunmethylated cytosine residue or a methylated cytosine residue. Forexample, the chemical reagent can be hydrazine, wherein the methodfurther includes contacting the hydrazine treated SOCS-1 gene sequencewith an agent that cleaves hydrazine modified cytosine residues toproduce fragments; separating the fragments according to molecularweight; and detecting a gap at a position known to contain a cytosineresidue in a SOCS-1 gene sequence, wherein the gap is indicative ofmethylation of a cytosine residue in the CpG dinucleotide in the SOCS-1gene of the test cell. The chemical reagent also can be sodiumbisulfite, wherein the method further includes exposing the sodiumbisulfite ion treated SOCS-1 gene sequence to alkaline conditions,whereby bisulfite modified cytosine residues are converted to uracilresidues; and detecting the amount or distribution of uracil residues inthe bisulfite ion treated SOCS-1 gene of the test cell, wherein adecrease in the amount or distribution of uracil residues in the SOCS-1gene from the test cell, as compared to the amount or distribution ofuracil residues in a corresponding bisulfite ion treated unmethylatedSOCS-1 gene following exposure to alkaline conditions, is indicative ofmethylation of cytosine residues in CpG dinucleotides in the SOCS-1 geneof the test cell.

The amount or distribution of uracil residues can be detected, forexample, by determining the nucleotide sequence of the bisulfite iontreated SOCS-1 gene sequence following exposure to alkaline conditions.The bisulfite ion treated SOCS-1 gene sequence can be determineddirectly, or can be amplified using an amplification primer pair, forexample, an amplification primer pair selected from SEQ ID NO:6 and SEQID NO:7; SEQ ID NO:12 and SEQ ID NO:13; SEQ ID NO:14 and SEQ ID NO:15;and SEQ ID NO:16 and SEQ ID NO:17, and the nucleotide sequence of theamplification product can be determined.

The amount or distribution of uracil residues also can be detected bycontacting the sodium bisulfite treated SOCS-1 gene sequence, followingexposure to alkaline conditions, with an oligonucleotide thatselectively hybridizes to a SOCS-1 gene sequence containing uracilresidues, for example, an oligonucleotide as set forth in SEQ ID NO:2,and detecting selective hybridization of the oligonucleotide. The amountor distribution of uracil residues also can be detected by contactingthe SOCS-1 gene sequence with a methylation-specific amplificationprimer pair comprising a forward primer and a reverse primer underconditions suitable for amplification, wherein at least one of theforward primer and the reverse primer comprises an oligonucleotide thatselectively hybridizes to a SOCS-1 gene sequence containing uracilresidues, for example, the primer pair set forth as SEQ ID NO:2 and SEQID NO:3, whereby generation of an amplification product is indicative ofmethylation of cytosine residues in CpG dinucleotides in the SOCS-1 geneof the test cell, thereby identifying the test cell as a neoplasticcell; or by contacting the SOCS-1 gene sequence with anunmethylation-specific amplification primer pair comprising a forwardprimer and a reverse primer under conditions suitable for amplification,wherein both the forward primer and the reverse primer selectivelyhybridize to a SOCS-1 gene sequence containing cytosine residues, butnot to a SOCS-1 gene sequence containing uracil residues, for examplethe primer pair set forth as SEQ ID NO:4 and SEQ ID NO:5, wherebygeneration of an amplification product is indicative of a lack ofmethylation of cytosine residues in CpG dinucleotides in the SOCS-1 geneof the test cell, thereby identifying the test cell as a neoplasticcell.

Similarly, the amount or distribution of uracil residues can be detectedby contacting the SOCS-1 gene sequence with at least two amplificationprimer pairs, including a methylation-specific amplification primerpair, for example, SEQ ID NO:2 and SEQ ID NO:3, and anunmethylation-specific amplification primer pair, for example, SEQ IDNO:4 and SEQ ID NO:5, under conditions suitable for amplification,wherein an amplification product, if any, generated by themethylation-specific amplification primer pair has a first length, andwherein an amplification product, if any, generated by theunmethylation-specific amplification primer pair has a second length,which is different from the first length, whereby generation of anamplification product having the first length is indicative methylationof cytosine residues in CpG dinucleotides in the SOCS-1 gene of the testcell, thereby identifying the test cell as a neoplastic cell.

The present invention also relates to a method of reducing or inhibitingunregulated growth of a cell exhibiting methylation silencedtranscription of a SOCS/CIS gene. Such a method can be performed byproviding the cell exhibiting unregulated growth with a polypeptideencoded by the methylation-silenced SOCS/CIS gene, for example, byrestoring expression of the SOCS/CIS gene in the cell.

In one embodiment, restoring expression of the SOCS/CIS polypeptidecomprises contacting the cell with a demethylating agent such as5-aza-2′-deoxycytidine. Such contacting can be performed on a cell inculture by adding the demethylating agent to the cell culture medium.Such a cell in culture can be a cell of an established cell line, or canbe a cell, generally a population of cells, which can be a mixedpopulation of cells, that has been removed from a subject and is beingcontacted ex vivo, for example, to determine whether contact with theparticular demethylating agent can restore the SOCS/CIS gene expression,or to restore such SOCS/CIS gene expression, after which the cells,which can be, for example, bone marrow cells of an individual sufferingfrom multiple myeloma or an acute leukemia, are administered back intothe subject. Contacting the cell with the demethylating agent also canbe performed in vivo by administering the agent to a subject. Whereconvenient, the demethylating agent is administered at or near the siteof the cells exhibiting unregulated growth in the subject, or into ablood vessel in which the blood is flowing to the site of the targetcells.

In another embodiment, restoring expression of the SOCS/CIS polypeptideto a cell exhibiting unregulated growth is performed by introducing apolynucleotide encoding the SOCS/CIS polypeptide into the cell, wherebythe SOCS/CIS polypeptide is expressed from the polynucleotide. Forexample, where the cell is characterized by methylation-silencedtranscription of the SOCS-1 gene, the polynucleotide can comprise SEQ IDNO:1. The polynucleotide encoding the SOCS/CIS polypeptide can include,in addition to SOCS/CIS polypeptide coding sequence, operatively linkedtranscriptional regulatory elements, translational regulatory elements,and the like, which allow for expression of the encoded polypeptide atleast in the target cell. The polynucleotide can be introduced into acell in culture, for example, a cell ex vivo, or can be introduced intoa cell in vivo. In addition, the polynucleotide can be in the form of anaked DNA molecule, or can be formulated in a matrix that facilitatesentry of the polynucleotide into the particular cell, for example, aliposome, in which case the polynucleotide contains the requiredoperatively linked regulatory elements. The SOCS/CIS polynucleotide alsocan be contained in a vector, which can provide some or all of theregulatory elements required for expression of the encoded SOCS/CISpolypeptide. The vector can be any vector suitable for introducing apolynucleotide into the particular cell exhibiting unregulated growth,including, for example, a viral vector such as a viral vector derivedfrom a retrovirus or other lentivirus, an adenovirus, anadeno-associated virus, or a herpesvirus.

In still another embodiment, the SOCS/CIS polypeptide is provideddirectly to the cell exhibiting unregulated growth. The polypeptide canbe contacted with the cell in vitro under conditions that result insufficient permeability of the cell such that the polypeptide can crossthe cell membrane, or the polypeptide can be microinjected into thecells. Where the SOCS/CIS polypeptide is contacted with a cell in situin an organism, it can comprise a fusion protein, which includes, forexample, a peptide or polypeptide component that facilitates transportacross the cell membrane (e.g., a human immunodeficiency virus (HIV) TATprotein transduction domain), or can be formulated in a matrix thatfacilitates entry of the polypeptide into a cell.

The present invention also relates to a method for treating a cancerpatient, wherein cancer cells in the patient exhibit methylationsilenced SOCS-1 gene expression, by providing SOCS-1 polypeptide (SEQ IDNO:20) to the cancer cells, thereby inducing apoptosis of the cells.SOCS-1 polypeptide can be provided to the cells by contacting the cellswith a demethylating agent, for example, by administering thedemethylating agent to the subject in an amount sufficient to restoreSOCS-1 gene expression in the cancer cells, or by introducing apolynucleotide encoding SOCS-1, for example, the polynucleotide setforth as SEQ ID NO:1 or a polynucleotide encoding SEQ ID NO:20, into thecancer cells under conditions sufficient for expression of the encodedSOCS-1 polypeptide in the cancer cells. Where the polynucleotide isadministered to a subject, the polynucleotide can be contained in avector, particularly a vector derived from a virus that preferentiallyinfects the cells from which the cancer cells arose, for example, avector derived from a hepatitis vector where the cancer cells arehepatocellular carcinoma cells, or a vector derived from HIV where thecancer cells are T cell leukemia cells. The polynucleotide, which can becontained in a vector, also can be formulated with a matrix such as aliposome, which can be further modified to contain a receptor (orligand) on its surface, wherein the receptor (or ligand) canspecifically bind a cognate ligand (or receptor) expressed by the cancercells, for example, the liposome can contain on its surface antibodiessuch as anti-idiotype antibodies that specifically bind with antibodiesexpressed by plasma cells associated with a multiple myeloma. Thepolynucleotide also can comprise an operatively linked regulatoryelement that directs expression of the polynucleotide, particularly in atissue specific manner such that the polynucleotide is expressed only inthe target cells, for example, an I-fetoprotein promoter, which isactive in hepatocytes, including hepatocellular carcinoma cells; or aleukosialin (CD43) or leukocyte common antigen (LCA; CD45) promoter,which is active in leukocytes or hematopoietic cells, respectively.

The present invention further relates to a method for selecting atherapeutic strategy for treating a cancer patient by detectingmethylation-silenced transcription of a SOCS/CIS gene in cancer cells ofthe patient. Such a method can be performed by examining a samplesuspected of containing cancer cells from the patient for decreasedexpression of a SOCS/CIS gene product due to methylation-silencedtranscription, whereby detecting decreased expression of a SOCS/CIS geneproduct indicates selecting an agent that restores the SOCS/CIS geneproduct to the cancer cells as a component of the therapeutic strategy.

Upon determining that a cancer is associated with methylation-silencedtranscription of a SOCS/CIS gene, the agent selected for restoringSOCS/CIS gene product to the cell can be an agent such as ademethylating agent, which restores transcriptional activity of thesilenced SOCS/CIS gene, or an agent that modulates the activity of aneffector molecule downstream of the SOCS/CIS gene in a signaltransduction pathway, for example, an agent such as AG490, whichinhibits the activity of JAK2 kinase, which is downstream of andregulated by SOCS-1 in a signal transduction pathway. The agent to beselected also can be selected based on the particular SOCS/CIS gene thatis methylation-silenced. For example, where the cancer is associatedwith methylation-silenced SOCS-1 gene expression, the agent can be theSOCS-1 protein, or a polynucleotide encoding SOCS-1, such as thepolynucleotide set forth in SEQ ID NO:1, or a substantially similarpolynucleotide having, for example, one or more silent nucleotidechanges that change a codon to a degenerate codon encoding the sameamino acid.

The present invention also relates to a method of treating a subjectsuffering from hepatocellular carcinoma (HCC) or multiple myeloma (MM),wherein cells associated with the HCC or MM exhibit methylation silencedSOCS-1 gene expression, the method comprising administering an amount ofAG490 to the subject sufficient to induce apoptosis of the cellsassociated with the cancer.

The present invention further relates to an isolated oligonucleotide,which is useful as a probe or a primer for detecting a SOCS-1 genesequence, or in combination as amplification primer pairs for amplifyingall or a portion of a SOCS-1 gene sequence, including, for example, amethylated SOCS-1 gene sequence or an unmethylated SOCS-1 gene sequence.For example, the present invention provides an oligonucleotide selectedfrom any one of SEQ ID NOS:2 to 6 and 12 to 17, and further provides aplurality of such oligonucleotides, which includes at least two of theoligonucleotides set forth as SEQ ID NOS:2 to 6 and 12 to 17. Alsoprovided is an amplification primer pair, comprising a forward primerand a reverse primer such as those set forth as SEQ ID NO:2 and SEQ IDNO:3; SEQ ID NO: 4 and SEQ ID NO:5; SEQ ID NO:6 and SEQ ID NO:7; SEQ IDNO:12 and SEQ ID NO:13; SEQ ID NO:14 and SEQ ID NO:15; or SEQ ID NO:16and SEQ ID NO:17; as well as a plurality of such amplification primerpairs, comprising at least two primer pairs, including at least one ofthe primer pairs set forth as SEQ ID NO:2 and SEQ ID NO:3; SEQ ID NO: 4and SEQ ID NO:5; SEQ ID NO:6 and SEQ ID NO:7; SEQ ID NO:12 and SEQ IDNO:13; SEQ ID NO:14 and SEQ ID NO:15; or SEQ ID NO:16 and SEQ ID NO:17.

In one embodiment, the amplification primer pair, or a pluralitythereof, includes SEQ ID NO:2 and SEQ ID NO:3, which can specificallyamplify a methylated SOCS-1 gene sequence. In another embodiment, theamplification primer pair, or a plurality thereof, includes SEQ ID NO:4and SEQ ID NO:5, which can specifically amplify a unmethylated SOCS-1gene sequence. In still another embodiment, a plurality of primer pairs,which includes SEQ ID NOS:2 and 3, and SEQ ID NOS:4 and 5 is provided.

The present invention further relates to a kit, which contains reagentsuseful for practicing a method of the invention. As such, a kit of theinvention can contain, for example, an isolated oligonucleotide,comprising any one of SEQ ID NOS:2 to 6 and 12 to 17; at least twoisolated oligonucleotides selected from SEQ ID NOS:2 to 6 and 12 to 17;at least one amplification primer pair selected SEQ ID NO:2 and SEQ IDNO:3; SEQ ID NO: 4 and SEQ ID NO:5; SEQ ID NO:6 and SEQ ID NO:7; SEQ IDNO:12 and SEQ ID NO:13; SEQ ID NO:14 and SEQ ID NO:15; and SEQ ID NO:16and SEQ ID NO:17; an amplification primer pair comprising SEQ ID NO:2and SEQ ID NO:3; or an amplification primer pair comprising SEQ ID NO:4and SEQ ID NO:5.

In one embodiment, a kit of the invention contains a plurality ofoligonucleotides, including those having sequences as set forth in SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, wherein theoligonucleotides are useful as probes, or in combination asamplification primer pairs, for detecting methylated SOCS-1 genesequences, unmethylated SOCS-1 gene sequences, or both. In anotherembodiment, a kit of the invention contains an amplification primerpair, or a plurality thereof, wherein at least one oligonucleotide ofthe primer pair is selected from SEQ ID NOS:2 to 6 and 12 to 17. A kitof the invention can further include one or more amplification primerpairs selected to allow nested amplification of an amplification productgenerated using a first amplification primer pair of the kit. A kit ofthe invention also can contain reagents for performing an amplificationreaction; a reagent that modifies methylated cytosine residues; amethylation sensitive restriction endonuclease; or a combination of suchreagents.

DETAILED DESCRIPTION OF THE INVENTION

As disclosed herein unregulated cell growth as occurs in various cancershas been correlated with methylation-silenced transcription of asuppressor of cytokine signaling (SOCS)/cytokine-inducible SH2 protein(CIS) family member (SOCS/CIS) gene. The correlation ofmethylation-silenced SOCS/CIS gene expression and cancer is exemplifiedby the demonstration that methylation-silenced transcription of theSOCS-1 gene is associated with various types of cancer, includinghepatocellular carcinoma, multiple myeloma, and acute leukemias, andthat restoration of SOCS-1 gene expression is such cancer cells resultsin apoptosis of the cells. As such, the present invention providesmethods of diagnosing a cancer by detecting methylation-silencedtranscription of a SOCS/CIS gene, as well as methods of treating suchcancers by restoring the SOCS/CIS gene product to the cells.

The SOCS/CIS family of proteins has been implicated in the negativeregulation of several cytokine pathways, including, for example,interleukin-6 (IL-6). SOCS-1 specifically associates with Janus kinase(JAK) to switch off cytokine signaling via the JAK/signal transducer andactivator of transcription (STAT) pathways of transcriptional activation(Masuhara et al., Biophys. Res. Comm. 239:439-446, 1997; Hilton et al.,Proc. Natl. Acad. Sci. USA 95:114-119, 1998; Starr et al., Nature387:917-921, 1997, each of which is incorporated herein by reference).As disclosed herein, aberrant methylation in the CpG island of theSOCS-1 gene correlated with transcriptional silencing of SOCS-1 inhepatocellular carcinoma (HCC) cell lines, including an incidence ofaberrant methylation of 65% in 26 human primary HCC tumor samplesanalyzed (Example 1; see, also, Yoshikawa et al., Nat. Genet. 28:29-35,2001, which is incorporated herein by reference). In HCC cellsexhibiting methylation-silenced SOCS-1 gene expression, restoration ofSOCS-1 gene expression suppressed the growth rate andanchorage-independent growth of the cells, and suppressed constitutivelyactivated JAK2 expression. Moreover, the growth suppression was due toapoptosis of the cells, and was reproduced by treatment of the cellsAG490, a specific chemical inhibitor of JAK2 that reversed constitutivephosphorylation of STAT3 in SOCS-1 inactivated cells.

In view of the results observed for HCC cell lines and primary HCC tumorsamples, and further in view of role of IL-6 as a survival factor inmultiple myeloma (MM), methylation of the SOCS-1 gene CpG island wasexamined in MM cell lines and in primary MM samples. Aberrant SOCS-1methylation was detected in two IL-6-dependent MM cell lines, U266 andXG1, and, as for HCC cells, correlated with transcriptional silencing.Treatment of the MM cells with a demethylating agent,5-aza-2′-deoxycytidine (5-azacytidine), resulted in up-regulation ofSOCS-1 gene expression. Methylation-silenced transcription of SOCS-1 inthe MM cell lines correlated with greater sensitivity to the chemicalJAK inhibitor, AG490. Using methylation-specific PCR (MSP), SOCS-1hypermethylation was detected in 62.9% (23/35) of MM patient samples. Incomparison, analysis of malignant lymphomas of various histologic typesrevealed that SOCS-1 was hypermethylated in only 3.2% (2/62), and nomethylation of the SOCS-1 gene was detected in normal peripheral bloodleukocytes or bone marrow cells.

Since the JAK/STAT pathway has a crucial role in hematological cells,and is negatively regulated by SOCS-1, the methylation status of theSOCS-1 gene CpG island also was examined with respect to acuteleukemias. SOCS-1 gene methylation was detected in each of the types ofacute leukemia examined, including in samples from a de novo acutemyelogenous leukemia (AML), a secondary AML transformed frommyelodysplastic syndrome (MDS), a B cell acute lymphocytic leukemia(ALL), and a T cell ALL. The incidence of methylation was 31% for all ofthe samples examined, and was 50% for the MDS based AML. Constitutiveactivation of STAT1, STAT3 and STAT5 was detected in an AML cell line,and treatment with AG490 induced apoptosis, and resulted in decreasedSTAT1 and STAT3 tyrosine phosphorylation levels.

These results demonstrate that inactivation of SOCS-1 gene expressiondue to hypermethylation of the SOCS-1 gene correlated to the unregulatedgrowth characteristic of several different types of cancer, includinghepatocellular carcinoma, multiple myeloma, and acute leukemia. Inaddition, the results disclosed herein demonstrate that agents such asthe demethylating agent, 5-azacytidine, which restores SOCS-1 geneexpression in cancer cells, and the JAK inhibitor, AG490, whichcircumvents the lack of SOCS-1 activity, can induce apoptosis of thecancer cells. The high prevalence of the aberrant SOCS-1 methylation,and its growth suppression activity, demonstrates the importance of theconstitutive activation of the JAK/STAT pathway in various cancers, andindicates that SOCS-1 methylation status can be targeted for diagnosticand therapeutic strategies. Accordingly, the present invention providesmethods for diagnosis of a cancer associated with aberrant CpGmethylation in the SOCS-1 gene, methods for treating such cancers, andreagents for practicing such methods.

SOCS-1 is a member of the SOCS/cytokine-inducible SH2 protein (CIS)family of proteins (SOCS/CIS family). The SOCS/CIS family members have apoorly conserved N-terminal region, a central SH2 domain, which caninteract with phosphorylated tyrosine, and a SOCS box, which is a domainthat is conserved among at least 20 proteins but for which a functionhas not yet been described (Endo et al., Nature 387:921-924, 1997; Nakaet al., Nature 387:924-929, 1997). SOCS-1 is involved in a negativefeedback loop of cytokine signaling (see, for example, Masuhara et al.,supra, 1997; Hilton et al., supra, 1998; Starr et al., supra, 1997). Thehuman SOCS-1 gene is localized in chromosome 16p13.3, just downstream ofthe protamine gene cluster, and contains a single exon gene encoding the211 amino acid residue SOCS-1 protein (see, for example, Kramer et al.,J. Exp. Zool. 282:245-255, 1998, which is incorporated herein byreference; see, also, SEQ ID NO: 20). The SOCS-1 gene lies within alarge CpG island spanning about 2.5 kilobases (kb).

SOCS-1 expression renders cells unresponsive to IL-6 stimulation.Various cytokines, including, for example, IL-4, IL-13, interferon-γ(IFN-γ), leukemia inhibitory factor (LIF), and granulocyte-monocytecolony stimulating factor (GM-CSF), as well as IL-6, can induce SOCS-1gene expression in hematologic cells; SOCS-1 interacts specifically withJAK to turn off the cytokine-mediated signal transduction. For example,the SH2 domain of SOCS-1 can specifically bind to a JH1 domain of JAK2,inhibiting the phosphorylation of JAK2 and down-regulating the JAK/STATpathway (see Hilton et al., supra, 1998). SOCS-1 also inhibits thebiological effects of cytokines in vivo such that forced SOCS-1expression interrupts macrophage differentiation induced by IL-6, andsuppresses CD23 expression induced by IL-4 (see Losman et al., J.Immunol. 162:3770-3774, 1999). As disclosed herein, SOCS-1 expressionalso has a role in the loss of growth control characteristic of cancer.

Relationships between protein tyrosine phosphorylation and cancer havebeen demonstrated (see, for example, Ihle et al., Nature, 377:591-594,1995). For example, over-expression of a phosphotyrosine proteinphosphatase in NIH/3T3 cells and in v-erbB transformed cells inhibitedthe growth of both cell types in culture (Rampone it al., Int. J. Cancer51:652-656, 1992). PTEN is an example of a protein tyrosine phosphatasethat is mutated in various types of cancer, and reconstruction of thePTEN gene in the mutant cell lines resulted in significant growthsuppression (Furnari et al., Proc. Natl. Acad. Sci. USA94:12,479-12,484, 1997). Mice that were homozygous for mutations in the“motheaten” gene that result in defective hematopoietic cell phosphataseshowed CSF-1 independent proliferation of macrophage, and mice that wereheterozygous mutants showed an increased incidence of lymphoreticularneoplasia. A mutation in the EPOR gene resulted in expression of mutantprotein that hematopoietic cell phosphatase was unable to bind,resulting in induced prolonged activation of JAK2 (Klingmuller et al.,Cell 80:729-738, 1995).

Abnormalities of the JAK/STAT pathway have been associated with cancer(Ihle et al., supra, 1995; Garcia et al., Cell Growth Diff 8:1267-1276,1998). For example, a dominant gain-of-function mutation in a DrosophilaJAK (hop^(tum-1)), in which the Drosophila JAK protein wasphosphorylated to a greater extent than in the wild type, causedleukemia-like abnormalities (Luo et al., EMBO J. 14:1412-1420, 1995;Harrison et al., EMBO J. 14:2857-2865, 1995). In addition, astructurally abnormal JAK was detected in human cancers. For example, at(9; 12)(p24; p13) chromosomal translocation was found in T cellchildhood ALL, resulting in a fusion of the catalytic domain of the JAK2to an oligomerization domain of ETV6 and constitutive activation of itstyrosine kinase activity (Lacronique et al., Science 278:1309-1312,1997). Furthermore, a SH2 domain mutant of STAT3 can spontaneously formhomodimers and bind DNA, resulting in transcriptional activation, andimmortalized mouse fibroblasts transfected with the mutant STAT3acquired the ability to grow in soft agar and were tumorigenic in nudemice (Bromberg et al., Cell 98:295-303, 1999). In contrast, expressionof a STAT3 dominant-negative induced apoptosis in myeloma cells(Catlett-Facone et al., Immunity 10:105-115, 1999).

In addition to inducing SOCS-1 gene expression, IL-6 also plays animportant role in B cell growth and differentiation into plasma cells,and is the essential growth and survival factor for neoplastic cells inthe pathogenesis of multiple myeloma (Klein et al., Blood 85:863-872,1995; Hallek et al., Blood 91:3-21. 1998; Hirano et al., Oncogene19:2548-2556, 2000). IL-6 binds to a specific membrane receptor (IL-6R),and the IL-6/IL-6R complex induces the dimerization of the receptorsubunit gp 130. Subsequently, members of the JAK family, whichcross-phosphorylate each other and the cytoplasmic domains of thereceptors on tyrosine residues, become activated, thus providing dockingsites for latent STAT transcription factors, which, in turn, becomephosphorylated and dimerize before entering the nucleus and initiatingtranscription of target genes, including proliferative genes such asc-myc an cyclin D1, and anti-apoptotic genes such as pim-1 and bc1-x1(see, for example, Heinrich et al., Biochem. J. 334:297-314, 1998; Imadaet al., Mol. Immunol. 37:1-11, 2000). Under physiological conditions,this signaling is quickly attenuated, and the stimulation by thecytokine is thereby limited. A key component of this negative feedbackloop is SOCS-1, which down-regulates JAK/STAT effects by directlyinteracting with JAK.

There is increasing evidence for a role of the JAK/STAT pathway in thepathogenesis of different leukemias. Constitutive activation of severalSTAT proteins, particularly STAT1, STAT3 and STAT5 has been found indifferent types of leukemia (see, for example, Coffer et al., Oncogene,19:2511-2522, 2000; Ward et al., Blood 95:19-29, 2000; Lin et al.,Oncogene 19:2496-2504, 2000). For example, STAT3 (28%) and STAT5 (22%)are activated in AML (Xia et al., Cancer Res. 58:3173-3180, 1998). Inaddition, a number of defined genetic aberrations have been shown to beresponsible for activation of the JAK/STAT pathway, including the t(9;12)(p24; p13) chromosomal translocation as discussed above (Lacroniqueet al., supra, 1997).

SOCS family proteins function as negative regulators of JAK/STATsignaling. Eight members of the SOCS family have been identified,including SOCS-1 to SOCS-7 and CIS (cytokine-inducible SH2-containingprotein; Alexander et al., J. Leuk. Biol. 66:588-592, 1999; Nicola etal., Expt. Hematol. 28:1105-1112, 2000). Blocking of JAK activation bySOCS-1 results in inhibition of the JAK/STAT signal transductionpathway, thus suppressing signaling by a wide variety of factorsincluding, for example, IL-6, IL-4, LIF, oncostatin M, growth hormone,prolactin, thrombopoietin and interferons (see Greenhalgh and Hilton, J.Leuk. Biol. 70:348-356, 2001). As disclosed herein, methylation of theSOCS-1 gene results in transcriptional silencing of SOCS-1 geneexpression, thus leading to unregulated JAK/STAT signal transductionactivity in cancer cells, and unregulated growth of the cells (seeExamples 1 to 3).

The silencing of gene transcription associated with aberrant DNAmethylation of CpG dinucleotides in normally unmethylated gene promoterregions is the most widely studied epigenetic abnormality intumorigenesis. The binding of protein complexes consisting ofmethyl-CpG-binding domains, transcriptional co-repressors, chromatinremodeling proteins and histone deacetylases to hypermethylated DNAregions results in a transcriptionally repressed (silenced) chromatinstate. In eukaryotic cells, methylation of cytosine residues that areimmediately 5′ to a guanosine residue occurs predominantly in CG poorregions. In contrast, CpG islands generally remain unmethylated innormal cells, except during X chromosome inactivation and parentalspecific imprinting, where methylation of 5′ regulatory regions isassociated with transcriptional repression. De novo methylation of theretinoblastoma (Rb) gene has been demonstrated in a small fraction ofretinoblastomas (Sakai et al., Am. J. Hum. Genet. 48:880, 1991), andaberrant methylation of the VHL gene was found in a subset of sporadicrenal cell carcinomas (Herman et al., Proc. Natl. Acad. Sci. USA91:9700-9704, 1994). Expression of a tumor suppressor gene can also beabolished by de novo DNA methylation of a normally unmethylated 5′CpGisland (see, for example, Issa et al., Nature Genet. 7:536, 1994; Merloet al., Nature Med. 1:686, 1995; Herman et al., Cancer Res. 56:722,1996).

Aberrant methylation of promoter regions in CpG islands also has beenassociated with the development of cancer. In hematopoieticmalignancies, for example, hypermethylation of E-cadherin (Graff et al.,Cancer Res. 55:5195-5199, 1995), DAP-kinase (Katzenellenbogen et al.,Blood 93:4347-4353, 1999), and the cell cycle regulators p15^(INK4B) andp16^(INK4A), is associated with gene inactivation (Herman et al., CancerRes. 57:837-841 1997; Melki et al., Blood 95:3208-3213, 2000; Ng et al.,Clin. Canc. Res. 7:1724-1729, 2001). Transcriptional silencing due tohypermethylation also has been detected in the CDKN2A gene (Herman etal., Cancer Res. 55:4525-4530, 1995), MGMT (Esteller et al., Cancer Res.59:793-797, 1999), and MLH1 gene (Herman et al., Proc. Natl. Acad. Sci.USA 95:6870-6875, 1998).

Hypermethylation of a CpG island at chromosome position 17p13.3 has beenobserved in multiple common types of human cancers (Makos et al., Proc.Natl. Acad. Sci. USA 89:1929, 1992; Makos et al., Cancer Res. 53:2715,1993; Makos et al., Cancer Res. 53:2719, 1993), and coincides withtiming and frequency of 17p loss and p53 mutations in brain, colon, andrenal cancers. Silenced gene transcription associated withhypermethylation of the normally unmethylated promoter region CpGislands has been implicated as an alternative mechanism to mutations ofcoding regions for inactivation of tumor suppressor genes (Baylin etal., Cancer Cells 3:383, 1991; Jones and Buckley, Adv. Cancer Res.54:1-23, 1990). This change also has been associated with the loss ofexpression of VHL, a renal cancer tumor suppressor gene on 3p (Herman etal., supra, 1994), the estrogen receptor gene on 6q (Ottaviano et al.,Cancer Res. 54:2552, 1994), and the H19 gene on 11p (Steenman et al.,Nature Genetics, 7:433, 1994).

A DNA region, termed Spot7, demonstrated the highest aberrationincidence of up to 88% in restriction landmark genomic scanning (RLGS)analysis of HCC (Nagai et al., Cancer Res. 54:1545-1550, 1994). Spot7was localized to chromosome 16, where the aberrant DNA in the RLGSanalysis was most concentrated (Yoshikawa et al., Gene 197:129-135,1997). As disclosed herein, a Not I restriction endonuclease site in theSOCS-1 gene in the Spot7 region was methylated in HCC, and furtherexamination revealed that methylation silencing of the SOCS-1 gene, andloss of growth suppression activity by SOCS-1 protein, correlated withHCC, as well as other cancers, including acute leukemias and multiplemyeloma.

Accordingly, the present invention provides a method for identifying acell that exhibits, or is predisposed to exhibiting, unregulated growth,by detecting methylation of cytosine residues in CpG dinucleotides in aCpG island of a SOCS/CIS gene in a test cell, or an extract comprisingnucleic acid molecules of the test cell, wherein the SOCS/CIS genemethylation results in a reduced level of transcription of the gene inthe test cell as compared to a corresponding cell that exhibits normalregulated growth. As used herein, the term “methylation” or“hypermethylation”, when used in reference to a SOCS/CIS gene, meansthat cytosine residues of CpG dinucleotides in a CpG island associatedwith the gene are methylated at the 5′-position, i.e.,5′-methylcytosine. The terms are used interchangeably because thecytosine residues in a SOCS/CIS gene CpG island normally areunmethylated, and, therefore, any amount of cytosine methylation in aCpG island also can be considered hypermethylation.

A CpG island of SOCS/CIS gene is exemplified by the CpG island thatspans about 2.5 kilobases of chromosome 16, including the SOCS-1 gene,and further spanning about nucleotides 200 to 500 of the SOCS-1 codingsequence beginning with the ATG start codon (see SEQ ID NO:1). The term“methylation status” is used herein to refer to relative abundance,including the presence or absence, of methylated cytosine residues ofCpG dinucleotides in a CpG island. As such, a method of the inventionprovides a means to determine the methylation status of a SOCS/CIS genein a cell.

In general, the cytosine residues in a CpG island are not methylated ina transcriptionally active SOCS/CIS gene and, therefore, the detectionof methylated cytosine residues in a CpG island indicates that theSOCS/CIS gene is not being expressed. As such, reference herein to a“methylation-silenced” SOCS/CIS gene means that the gene is not beingtranscribed, or is being transcribed at a level that is decreased withrespect to the level of transcription of a corresponding unmethylatedSOCS/CIS gene. Methylation-silencing is detectable by progressivelydecreasing levels of transcriptional activity of a gene such as SOCS-1as methylation of cytosine residues in CpG dinucleotides of a CpG islandincreases to encompass about 15% to 20% of the cytosine residues, atwhich point transcription generally is completely silenced. Aconsequence of methylation-silenced SOCS/CIS gene expression is that acell containing the gene has reduced levels of, or completely lacks, theSOCS/CIS gene product such that a regulatory activity due to theSOCS/CIS gene product in the cell is reduced or absent. For example,methylation-silenced SOCS-1 gene expression in a cell results inconstitutive activation of the JAK2 kinase and, therefore, unregulatedgrowth of the cell, due to an insufficient amount of SOCS-1 protein inthe cell to down-regulate JAK2 activity.

A method of the invention requires, in part, a comparison of SOCS/CISgene CpG island methylation in a test cell or sample with the level ofmethylation, if any, of a corresponding SOCS/CIS gene in a correspondingcell exhibiting regulated growth. As used herein, the term“corresponding” means a reference material, with which a test materialis being compared. Generally, the reference material provides a controlor standard with which the test material is compared. For example,reference to a corresponding unmethylated SOCS/CIS gene, with respect toa SOCS/CIS gene being examined for methylation status, means that theunmethylated SOCS/CIS gene is the same type of gene as the SOCS/CIS genebeing examined for methylation status, e.g., the test gene and thecorresponding unmethylated gene are both human SOCS-1 genes, or are bothmurine CISH genes, etc. Reference to a corresponding cell exhibitingregulated growth, with respect to a test cell, generally refers to anormal cell, i.e., a cell that has a cell cycle and growth patterncharacteristic of a population of such cells in a healthy individual,for example, a normal hepatocyte, where the test cell is suspected ofbeing a HCC cell, or a normal B cell, where the test cell is suspectedof being a B cell leukemia cell. Generally, a cell that exhibitsregulated growth survives for a period of time, after which apoptosis isinduced resulting in programmed death of the cell.

In general, a corresponding SOCS/CIS gene or a corresponding cellexhibiting regulated growth will be known to those in the art. Forexample, it will be recognized that a particular SOCS/CIS gene in aparticular cell type such as a SOCS-1 gene in a hepatocyte will have alevel and/or pattern of methylation, if any, that is characteristic forthat gene in the particular cell type, and that such methylation statuscan be determined by examining the SOCS/CIS gene sequence, using methodsas disclosed herein, in a statistically significant number ofhepatocytes from individuals of believed to be normal and healthyrepresentatives of a population. Similarly, the growth characteristicsof a statistically significant number of cells from a healthy individualor from individuals of a population can be examined to determine theparameters of normal regulated cell growth for the particular cell type.Such parameters can include, for example, the ability to grow (or notgrow) in soft agar, the susceptibility of the cells to contactinhibition of growth, the number of divisions the cells can undergounder defined conditions in a tissue culture medium, and theresponsiveness of the cells to a growth factor, hormone, or the like.

A method of the invention provides a means to identify a cell thatexhibits, or is predisposed to exhibiting unregulated growth. As usedherein, reference to a “cell exhibiting unregulated growth” means a cellthat has a growth characteristic that is different from that of acorresponding cell that exhibits regulated growth. Reference to a “cellpredisposed to exhibiting unregulated growth” means a cell that appearsto have the same growth characteristics in situ as that of acorresponding cell in situ, but that exhibits unregulated growth whenexamined under appropriate conditions, for example, in a soft agarassay, wherein the cell grows, or in a monolayer assay, wherein the celldoes not exhibit contact inhibition of growth. Such cells can beidentified by detecting methylation-silenced SOCS/CIS gene expressionaccording to a method of the invention.

The cell exhibiting, or predisposed to exhibiting, unregulated growthgenerally, but not necessarily, is a neoplastic cell, which can be apremalignant cell having a low level of CpG island methylation (e.g.,about 5% to 10%) and, therefore, a reduced level of expression of aSOCS/CIS gene product, or a malignant cell, i.e., a cancer cell, inwhich about 20% or more of the cytosine residues in the CpG island aremethylated and, therefore, transcription is completely inhibited. Suchcells, which exhibit methylation-silenced SOCS/CIS gene expression, areexemplified herein by hepatocellular carcinoma cells, multiple myelomacells, and various types of acute leukemia cells, each of which exhibitmethylation-silenced SOCS-1 gene expression. In addition, such cells caninclude ovarian carcinoma cells, breast carcinoma cells, pancreaticcancer cells, glioblastoma cells, lung cancer cells, melanoma cells, andthe like.

A method of the invention is practiced using a sample comprising a testcell, or an extract of the test cell that includes nucleic acidmolecules of the cell, particularly genomic DNA, including all or aportion comprising the CpG island of a SOCS/CIS gene that is to beexamined for methylation status. Generally, the test cell is a cell thatis suspected of being a cell that exhibits unregulated growth, forexample, a biopsy sample of suspicious lesion, or is a cell that is (orwas) in proximity to a premalignant or malignant cell, for example, cellsamples taken at one or few places outside of the region of a suspiciouslesion, such test cell providing an indication, for example, of theextent to which a surgical procedure should be performed, or a cellsample taken from a surgical margin, such test cells being useful fordetermining whether a cancer has been completely removed, or fordetermining whether a cancer has recurred.

A test cell examined according to a method of the invention also can bea primary cell that has been obtained from a subject and placed inculture, for example, for the purpose of establishing a primary cellculture that exhibits substantially the same growth characteristics asthe cells from which the culture was established, or for the purpose oftreating and/or expanding the cells for readministration to the subject.For example, bone marrow cells can be obtained from a cancer patientsuffering from multiple myeloma or from an acute leukemia, wherein thecells exhibit methylation-silenced SOCS/CIS gene expression. The cellsbe treated in culture using an agent that restores the SOCS/CIS geneexpression (see below), optionally can be expanded in culture, then canbe administered back to the subject.

A test cell can be obtained from a subject in any way typically used inclinical setting for obtaining a sample containing the cells. Forexample, the test cells (or a sample comprising the test cells) can beobtained by a biopsy procedure such as needle biopsy of an organ ortissue containing the cells to be tested. As such, the test cells can beobtained from a liver sample, a bone marrow sample, a skin sample, alymph node sample, a kidney sample, a lung sample, a muscle sample, abone sample, a brain sample, or the like. The test cell also can be acomponent of a biological fluid, for example, blood, lymph,cerebrospinal fluid, saliva, sputum, stool, urine, or ejaculate. Ifappropriate, the test cells also can be obtained by lavage, for example,for obtaining test cells from uterus, the abdominal cavity, or the like,or using an aspiration procedure, for example, for obtaining a bonemarrow sample.

A method of the invention also can be practiced using an extract of atest cell, wherein the extract includes nucleic acid molecules of thetest cell, particularly genomic DNA, including all or a CpG islandcontaining portion of a SOCS/CIS gene to be examined. The extract can bea crude extract comprising, for example, a freeze-thawed sample of atissue containing the test cells; can comprise partially purifiedgenomic DNA, which can include, for example, components of the nuclearmatrix; or can comprise substantially purified genomic DNA, which isobtained, for example, following treatment with a protease and alcoholprecipitation. In certain embodiments, the test cell also can be acomponent of a histologic sample that is embedded in paraffin.

Methylation of a CpG dinucleotide in a CpG island of a SOCS/CIS gene canbe detected using any of various well known methods for detecting CpGmethylation of a nucleic acid molecule. Such methods include contactingthe SOCS/CIS gene sequence with one or a series of chemical reagentsthat selectively modify either unmethylated cytosine residues ormethylated cytosine residues, but not both, such that the presence orabsence of the modification can be detected; contacting the SOCS/CISgene sequence with a methylation sensitive restriction endonuclease,which has a recognition site that includes a CpG dinucleotide, and thatcleaves a recognition site either having a methylated cytosine residueof the CpG or lacking a methylated cytosine residue of the CpG, but notboth, such that the presence or absence of cleavage of the sequence canbe detected; or contacting the SOCS/CIS gene sequence with anoligonucleotide probe, primer, or amplification primer pair thatspecifically hybridizes to the SOCS/CIS gene sequence and allows adetermination to made as to whether the CpG methylation is present.Examples of such methods are provided herein, and modifications andvariations on such methods are well known in the art.

In one embodiment, methylation of CpG dinucleotides in a CpG island of aSOCS/CIS gene sequence can be detected by contacting a nucleic acidmolecule, which includes all or a portion of a CpG island of theSOCS/CIS gene sequence, with a methylation sensitive restrictionendonuclease for which the SOCS/CIS gene contains the restrictionendonuclease recognition site. The methylation sensitive restrictionendonuclease can be one that cleaves a recognition site containing amethylated cytosine residue of a CpG dinucleotide, for example, arestriction endonuclease such as Acc III, Ban I, BstN I, Msp I, or XmaI, whereby cleavage of the nucleic acid molecule indicates that SOCS/CISgene in the test cell is methylated. Alternatively, the methylationsensitive restriction endonuclease can be one that cleaves a recognitionsite containing a CpG dinucleotide only when the cytosine residue of theCpG dinucleotide is unmethylated, for example, a restrictionendonuclease such as Acc II, Ava I, BssH II, BstU I, Hpa II, or Not I,whereby a lack of cleavage of the nucleic acid molecule indicates thatSOCS/CIS gene in the test cell is methylated.

The presence or absence of cleavage of the SOCS/CIS gene sequence by themethylation sensitive restriction endonuclease can be identified usingany method useful for detecting the length or continuity of apolynucleotide sequence. For example, cleavage of the SOCS/CIS gene canbe detected by Southern blot analysis, which allows mapping of thecleavage site, or using any other electrophoretic method orchromatographic method that separates nucleic acid molecules on thebasis of relative size, charge, or a combination thereof. Cleavage of aSOCS/CIS gene also can be detected using an oligonucleotide ligationassay, wherein, following contact with the restriction endonuclease, afirst oligonucleotide that selectively hybridizes upstream of andadjacent to a restriction endonuclease cleavage site and a secondoligonucleotide that selectively hybridizes downstream of and adjacentto the cleavage site are contacted with the SOCS/CIS gene sequence, andfurther contacted with a ligase such that, in the absence of cleavagethe oligonucleotides are adjacent to each other and can be ligatedtogether, whereas, in the absence of cleavage, ligation does not occur.By determining the size or other relevant parameter of theoligonucleotides following the ligation reaction, ligatedoligonucleotides can be distinguished from unligated oligonucleotides,thereby providing an indication of restriction endonuclease activity.

In another embodiment, methylation of a CpG dinucleotide in a CpG islandof a SOCS/CIS gene is detected by contacting a nucleic acid moleculecomprising the SOCS/CIS gene of the test cell with a chemical reagentthat selectively modifies either an unmethylated cytosine residue or amethylated cytosine residue, and detecting a product generated due tocontact with the reagent, wherein the product is indicative of themethylation status of CpG dinucleotides in the CpG island of theSOCS/CIS gene sequence. In one aspect of this embodiment, the SOCS/CISgene sequence is contacted with hydrazine, which modifies cytosineresidues, but not methylated cytosine residues, then the hydrazinetreated SOCS/CIS gene sequence is contacted with a reagent such aspiperidine, which cleaves the nucleic acid molecule at hydrazinemodified cytosine residues, thereby generating a product comprisingfragments. By separating the fragments according to molecular weight,using, for example, an electrophoretic, chromatographic, or massspectrographic method, and comparing the separation pattern with that ofa similarly treated unmethylated SOCS/CIS gene sequence, gaps will beapparent at positions in the test SOCS/CIS containing methylatedcytosine residues. As such, the presence of gaps is indicative ofmethylation of a cytosine residue in the CpG dinucleotide in theSOCS/CIS gene of the test cell.

In another aspect of this embodiment, the SOCS/CIS gene sequence iscontacted with a chemical reagent comprising bisulfite ions, forexample, sodium bisulfite, which converts unmethylated cytosine residuesto bisulfite modified cytosine residues, then the bisulfite ion treatedSOCS/CIS gene sequence is exposed to alkaline conditions, which convertbisulfite modified cytosine residues to uracil residues. Sodiumbisulfite reacts readily with the 5,6-double bond of cytosine (butpoorly with methylated cytosine) to form a sulfonated cytosine reactionintermediate that is susceptible to deamination, giving rise to asulfonated uracil. As such, the sulfonate group can be removed byexposure to alkaline conditions, resulting in the formation of uracil.The DNA then can amplified, for example, by PCR, and sequenced todetermine the methylation status of all CpG sites. Uracil is recognizedas a thymine by Taq polymerase and, upon PCR, the resultant productcontains cytosine only at the position where 5-methylcytosine waspresent in the starting template DNA. By comparing the amount ordistribution of uracil residues in the bisulfite ion treated SOCS/CISgene of the test cell with a similarly treated unmethylated SOCS/CISgene sequence, detection of a decrease in the amount or distribution ofuracil residues in the SOCS/CIS gene from the test cell is indicative ofmethylation of cytosine residues in CpG dinucleotides in the SOCS/CISgene of the test cell. The amount or distribution of uracil residuesalso can be detected by contacting the bisulfite ion treated SOCS/CISgene sequence, following exposure to alkaline conditions, with anoligonucleotide that selectively hybridizes to a SOCS/CIS gene sequencethat either contains uracil residues or that lacks uracil residues, butnot both, and detecting selective hybridization (or the absence thereof)of the oligonucleotide.

As used herein, the term “selective hybridization” or “selectivelyhybridize” refers to an interaction of two nucleic acid molecules thatoccurs and is stable under moderately stringent or highly stringentconditions. As such, selective hybridization preferentially occurs, forexample, between an oligonucleotide and a target nucleic acid molecule,and not substantially between the oligonucleotide and a nucleic acidmolecule other than the target nucleic acid molecule, including not withnucleic acid molecules encoding related but different members of afamily of proteins such as member of the SOCS/CIS family of protein.Generally, an oligonucleotide useful as a probe or primer thatselectively hybridizes to a target nucleic acid molecule is at leastabout 12 nucleotide in length, generally at least about 13 to 15nucleotides in length, and usually at least about 18 to 20 nucleotidesin length, or more. Examples of oligonucleotides useful in practicingthe methods of the invention are disclosed herein as SEQ ID NOS:2 to 7and 12 to 17, such oligonucleotides also being useful for identifyingadditional oligonucleotides that can selectively hybridize to otherspecific SOCS/CIS family member gene sequences and, therefore, be usedfor practicing the methods of the invention.

Conditions that allow for selective hybridization can be determinedempirically, or can be estimated based, for example, on the relativeGC:AT (or GC:AU) content of the hybridizing oligonucleotide and thetarget nucleic acid molecule, the length of the hybridizingoligonucleotide, and the number, if any, of mismatches between theoligonucleotide and sequence to which it is to hybridize (see, forexample, Sambrook et al., “Molecular Cloning”: A laboratory manual (ColdSpring Harbor Laboratory Press 1989)). As such, the conditions used toachieve a particular level of stringency will vary, depending on thenature of the hybridizing nucleic acid molecules. An additionalconsideration is whether one of the nucleic acids is immobilized, forexample, on a filter. An example of progressively higher stringencyconditions is as follows: 2×SSC/0.1% SDS at about room temperature(hybridization conditions); 0.2×SSC/0.1% SDS at about room temperature(low stringency conditions); 0.2×SSC/0.1% SDS at about 42° C. (moderatestringency conditions); and 0.1×SSC at about 68° C. (high stringencyconditions). Hybridization and/or washing can be carried out using onlyone of these conditions, for example, high stringency conditions, oreach of the conditions can be used, for example, for 10 to 15 minuteseach, in the order listed above, repeating any or all of the stepslisted.

Selective hybridization of an oligonucleotide with a target SOCS/CISgene sequence can be detected, for example, by performing the methodusing an oligonucleotide that includes a detectable label. Thedetectable label can be any molecule that conveniently can be linked tothe oligonucleotide and detected using readily available equipment. Forexample, the detectable label can be a fluorescent compound such a Cy3,Cy5, Fam, fluorescein, rhodamine, or a green fluorescent protein orenhanced or modified form thereof; a radionuclide such as sulfur-35,technicium-99, phosphorus-32, tritium or iodine-125; a paramagnetic spinlabel such as carbon-13, Gd-157, Mn-55, Dy-162, Cr-52, or Fe-56; aluminescent compound such as an aequorin; a chemiluminescent compound; ametal chelate; an enzyme such as luciferase or θ-galactosidase, or asubstrate for an enzyme; or a receptor or a ligand for a receptor, forexample, biotin. The means for detecting the detectable label will beselected based on the characteristics of the label, as will the meansfor linking the label to an oligonucleotide (see, for example,Hermanson, “Bioconjugate Techniques” (Academic Press 1996), which isincorporated herein by reference).

Selective hybridization also can be detected, for example, by utilizingthe oligonucleotide as a substrate for a primer extension reaction,further contacting the sample with deoxyribonucleotides (dNTPs),including, if desired, a detectable dNTP (e.g., a fluorescently labeleddNTP, a digoxigenin labeled dNTP, or a biotin labeled dNTP), and a DNAdependent DNA polymerase under conditions sufficient for the primerextension reaction to proceed, and detecting a product of the primerextension reaction. Conditions for performing a primer extensionreaction are well known in the art (see, for example, Sambrook et al.,supra, 1989).

The amount or distribution of uracil residues in a bisulfite ion treatedSOCS/CIS gene sequence following exposure to alkaline conditions alsocan be detected using an amplification reaction such as PCR. Anamplification reaction is performed under conditions that allowselective hybridization of primers to the target nucleic acid molecule.Generally, the reaction is performed in a buffered aqueous solution, atabout pH 7-9, usually about pH 8. In addition, the reaction generally isperformed in a molar excess of primers to target nucleic acid molecule,for example, at a ratio of about 100:1 primer:genomic DNA. Where theamount of the target nucleic acid molecule in a sample is not known, forexample, in a diagnostic procedure using a biological sample, a range ofprimer amounts can be used in samples run in parallel, althoughgenerally even the addition of a small amount of primers will result ina sufficient molar excess such that the amplification reaction canproceed.

The deoxyribonucleoside triphosphates, dATP, dCTP, dGTP, and dTTP, canbe added to the synthesis mixture either separately or as a mixture,which can further include the primers, in adequate amounts and theresulting solution is heated to about 90°-100° C. from about 1 to 10minutes, preferably from 1 to 4 minutes. After this heating period, thesolution is allowed to cool to room temperature, which is preferable forthe primer hybridization. To the cooled mixture is added an appropriateagent for effecting the primer extension reaction, generally apolymerase, and the reaction is allowed to occur under conditions asdisclosed herein (see Example 1) or otherwise known in the art. Wherethe polymerase is heat stable, it can be added together with the otherreagents. The polymerase can be any enzyme useful for directing thesynthesis of primer extension products, including, for example, E. coliDNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNApolymerase, other available DNA polymerases, polymerase muteins, reversetranscriptase, and other enzymes, including heat-stable enzymes, as arewell known in the art and commercially available. The amplificationproducts can be identified as methylated or non-methylated by asequencing method, oligomer restriction (Saiki et al., BioTechnology3:1008-1012, 1985), allele-specific oligonucleotide probe analysis(Conner et al., Proc. Natl. Acad. Sci. USA 80:278, 1983),oligonucleotide ligation assays (Landegren et al., Science 241:1077,1988), and the like (see, also, Landegren et al., Science 242:229-237,1988).

In one embodiment, the amplification is performed by contacting theSOCS/CIS gene sequence with an amplification primer pair comprising aforward primer and a reverse primer under conditions suitable foramplification, wherein at least one primer of the primer pair comprisesan oligonucleotide that selectively hybridizes to a SOCS/CIS genesequence containing uracil residues, whereby generation of anamplification product is indicative of methylation of cytosine residuesin CpG dinucleotides in the SOCS/CIS gene of the test cell. In anotherembodiment, the amplification reaction is performed by contacting theSOCS/CIS gene sequence with an amplification primer pair comprising aforward primer and a reverse primer under conditions suitable foramplification, wherein both primers of the primer pair selectivelyhybridize to a SOCS/CIS gene sequence containing cytosine residues, butnot to a SOCS/CIS gene sequence containing uracil residues, wherebygeneration of an amplification product is indicative of a lack ofmethylation of cytosine residues in CpG dinucleotides in the SOCS/CISgene of the test cell.

In still another embodiment, a methylation-specific amplificationreaction such as methylation-specific PCR (MSP) is used alone, or incombination with bisulfite treatment, to detect the methylation statusof a nucleic acid molecule (see U.S. Pat. Nos. 6,265,171; 6,200,756; and6,017,704, each of which is incorporated herein by reference; see, also,Example 1). MSP is a particularly sensitive method that allows detectionof low numbers of methylated alleles and the use of small amounts of anucleic acid sample, including paraffin-embedded materials, and also canbe conveniently adapted to a multiplex analysis, including, for example,simultaneous detection of unmethylated and methylated products in asingle sample, thus providing an internal control.

The amplification primer pairs used in an MSP reaction are designed tospecifically distinguish between bisulfite untreated or unmodified DNA,and methylated and unmethylated DNA. MSP primer pairs for theunmethylated DNA (unmethylation-specific primer pair) generally have athymidine residue in the 3′-CpG pair to distinguish it from the cytosineresidue retained in methylated DNA, and the complement is designed forthe antisense primer. MSP primer pairs usually contain relatively fewcytosine or guanine residues in the sequence because cytosine is absentin the sense (forward) primer and the guanine is absent in the antisense(reverse) primer; cytosine becomes modified to uracil, which isamplified as thymidine in the amplification product. An MSP(unmethylation-specific) primer pair is exemplified herein by the primerpair set forth as SEQ ID NOS:4 and 5, and a methylation-specific primerpair is exemplified herein by the primer pair set forth as SEQ ID NOS:2and 3. In view of the exemplified methylation-specific andunmethylation-specific primer pairs, and of the above description fordesigning such primer pairs, it will be recognized that additionalmethylation-specific and unmethylation-specific primer pairs useful foramplification of a methylated or an unmethylated SOCS-1 gene sequence(see SEQ ID NO:1; see, also, GenBank Acc. No. U15422), as well as forother SOCS/CIS gene sequences, which are well known in the art, can bereadily made.

Accordingly, in one aspect, MSP is used for detecting the amount ordistribution of uracil residues in a bisulfite ion treated SOCS/CIS genefollowing alkaline treatment. Such a method can be performed bycontacting the SOCS/CIS gene sequence with a first amplification primerpair and a second amplification primer pair under conditions suitablefor amplification, wherein the first amplification primer pair comprisesa forward primer and a reverse primer, and at least one primer of thefirst primer pair comprises an oligonucleotide that selectivelyhybridizes to a SOCS/CIS gene sequence containing uracil residues, andwherein the second amplification primer pair comprises a forward primerand a reverse primer, and both primers of the second primer pairselectively hybridize to a SOCS/CIS gene sequence containing cytosineresidues, but not to a SOCS/CIS gene sequence containing uracilresidues, and wherein an amplification product, if any, generated by thefirst primer pair has a first length, and an amplification product, ifany, generated by the second primer pair has a second length, which isdifferent from the first length, whereby the length of the amplificationproducts is indicative of the amount or distribution of uracil residuesand, therefore, of methylation of cytosine residues in CpG dinucleotidesin the SOCS/CIS gene of the test cell.

The methods of the invention are exemplified herein with respect toidentifying a neoplastic cell, which exhibits unregulated growth,wherein the method comprises detecting methylation of a cytosine residueof a CpG dinucleotide in a CpG island of a suppressor of cytokinesignaling-1 (SOCS-1) gene in a sample comprising a test cell, or anextract thereof, whereby methylation of the SOCS-1 gene results in areduced level of transcription and, therefore, expression of the SOCS-1gene product in the test cell as compared to a corresponding normalcell. The neoplastic cell can be a premalignant cell or a cancer cell,for example, a hepatocellular carcinoma cell, a multiple myeloma cell,or an acute leukemia cell. The sample to be examined can be obtainedfrom a subject such as a human subject, a domesticated animal, a farmanimal, or the like, and can contain a cell suspected of being aneoplastic cell, or can contain nucleic acid molecules including aSOCS-1 gene sequence or a portion thereof comprising a CpG island. Assuch, the sample can be an organ sample, a tissue sample, or a cellsample, for example, a liver sample, a skin sample, a lymph node sample,a kidney sample, a lung sample, a muscle sample, a bone sample, or abrain sample; or can be a sample of a biological fluid, for example,bone marrow, blood, serum, lymph, cerebrospinal fluid, saliva, sputum,stool, urine, or ejaculate.

Methylation of a CpG dinucleotide in a SOCS-1 gene can be detected bycontacting a nucleic acid molecule comprising the SOCS-1 gene of thetest cell with a methylation sensitive restriction endonuclease thatspecifically binds a recognition site containing a CpG dinucleotide, andthat either cleaves or does not cleave the recognition site depending onthe methylation status of the cytosine residue of CpG dinucleotide.Methylation of a CpG dinucleotide in a SOCS-1 gene also can be detectedby contacting a nucleic acid molecule comprising the SOCS-1 gene of thetest cell with a chemical reagent such as hydrazine or sodium bisulfiteand detecting the reaction products as discussed above.

Where the SOCS-1 gene sequence is treated using the bisulfite method,the amount or distribution of uracil residues then can be detected, forexample, by determining the nucleotide sequence of the bisulfite iontreated SOCS-1 gene sequence following exposure to alkaline conditions.The bisulfite ion treated SOCS-1 gene sequence can be determineddirectly, or can be amplified using an amplification primer pair, forexample, an amplification primer pair selected from SEQ ID NO:6 and SEQID NO:7; SEQ ID NO:12 and SEQ ID NO:13; SEQ ID NO:14 and SEQ ID NO:15;and SEQ ID NO:16 and SEQ ID NO:17, and the nucleotide sequence of theamplification product can be determined.

The amount or distribution of uracil residues also can be detected bycontacting the sodium bisulfite treated SOCS-1 gene sequence, followingexposure to alkaline conditions, with an oligonucleotide thatselectively hybridizes to a SOCS-1 gene sequence containing uracilresidues, for example, an oligonucleotide as set forth in SEQ ID NO:2,and detecting selective hybridization of the oligonucleotide; or bycontacting the SOCS-1 gene sequence with an amplification primer pairincluding a methylation specific primer pair such as the primer pair setforth as SEQ ID NO:2 and SEQ ID NO:3, whereby generation of anamplification product is indicative of methylation of cytosine residuesin CpG dinucleotides in the SOCS-1 gene of the test cell, or with anunmethylation specific amplification primer pair such as the primer pairset forth as SEQ ID NO:4 and SEQ ID NO:5, whereby generation of anamplification product is indicative of a lack of methylation of cytosineresidues in CpG dinucleotides in the SOCS-1 gene of the test cell,thereby identifying the test cell as a neoplastic cell.

In one embodiment, the amplification reaction comprises a multiplexanalysis, which can be performed, for example, by contacting the SOCS-1gene sequence with at least two amplification primer pairs, including amethylation-specific amplification primer pair such as the primer pairset forth as SEQ ID NO:2 and SEQ ID NO:3, and an unmethylation-specificamplification primer pair such as the primer pair set forth as SEQ IDNO:4 and SEQ ID NO:5, under conditions suitable for amplification. Themethylation-specific and unmethylation-specific primer pairs areselected such that an amplification product, if any, generated by themethylation-specific amplification primer pair has a first length, andan amplification product, if any, generated by theunmethylation-specific amplification primer pair has a second length,which is different from the first length. As such, generation of anamplification product having the first length is indicative ofmethylation of cytosine residues in CpG dinucleotides in the SOCS-1 geneof the test cell, thereby identifying the test cell as a neoplasticcell, whereas generation of an amplification product having the secondlength is indicative of a lack of methylation of cytosine residues inCpG dinucleotides in the SOCS-1 gene of the test cell, therebyidentifying that the test cell is not a neoplastic cell.

The diagnostic methods of the invention are particularly adaptable tobeing performed in a high throughput format, wherein a plurality of testcells, or extracts of the test cells, or test cells and extracts thereofcan be examined in parallel, preferably under automated orsemi-automated conditions. Generally, the reactions in a high throughputassay are performed on a solid support such as microchip, a glass slide,a bead, or the like, wherein the individual samples are substantiallyisolated from each other. In addition, the samples generally arearranged in an array or other reproducible pattern, such that eachsample can be assigned an address (i.e., a position on the array), thusfacilitating identification of the source of the sample. An additionaladvantage of arranging the samples in an array, particularly anaddressable array, is that an automated system can be used for adding orremoving reagents from one or more of the samples at various times, orfor adding different reagents to particular samples. In addition to theconvenience of examining multiple samples at the same time, such highthroughput assays provide a means for examining duplicate, triplicate,or more aliquots of a single sample, thus increasing the validity of theresults obtained, and for examining control samples under the sameconditions as the test samples, thus providing an internal standard forcomparing results from different assays.

The present invention also provides reagents useful for practicing thediagnostic methods of the invention, including, for example, an isolatedoligonucleotide, which is useful as a probe or a primer for detectingmethylation (or the absence thereof) of a SOCS/CIS gene sequence, or incombination as amplification primer pairs for amplifying all or aportion of a SOCS/CIS gene sequence, particularly all or a portion of aCpG island of the gene, including a methylated SOCS-1 gene sequence oran unmethylated SOCS-1 gene sequence. Oligonucleotides useful fordetecting SOCS-1 gene methylation are designed to be of sufficientlength and having an appropriate sequence such that they can selectivelyhybridize to a target nucleic acid sequence, particularly a nucleotidesequence of a SOCS/CIS gene that comprises, or is upstream or downstreamof a CpG island of the SOCS/CIS gene. Where the oligonucleotide is to beused as a substrate for a primer extension reaction, including anamplification reaction, the oligonucleotide is designed to allowspecific initiation and extension by a polymerase.

As used herein, the term “oligonucleotide” or “polynucleotide” is usedbroadly to mean a sequence of two or more deoxyribonucleotides orribonucleotides that are linked together by a phosphodiester bond. Forconvenience of discussion, the term “oligonucleotide” is used herein torefer to a polynucleotide that is used as a probe or primer, whereas theterm “polynucleotide” is used more broadly to encompass any sequence oftwo or more nucleotides, including an oligonucleotide. As such, theterms include RNA and DNA, which can be a gene or a portion thereof, acDNA, a synthetic polydeoxyribonucleic acid sequence, or the like.Generally, an oligonucleotide or polynucleotide can be single strandedor double stranded, as well as a DNA/RNA hybrid, although it will berecognized that the strands of a double stranded oligonucleotide that isto be used as a probe or primer will be separated, for example, byheating a solution containing the oligonucleotide above the meltingtemperature of the particular oligonucleotide.

The terms “oligonucleotide” and “polynucleotide” as used herein includenaturally occurring nucleic acid molecules, which can be isolated from acell, as well as fragments thereof as produced, for example, by arestriction endonuclease digestion, and synthetic molecules, which canbe prepared, for example, by methods of chemical synthesis or byenzymatic methods such as by PCR. In various embodiments, anoligonucleotide or polynucleotide of the invention can containnucleoside or nucleotide analogs, or a backbone bond other than aphosphodiester bond, for example, a thiodiester bond, a phosphorothioatebond, a peptide-like bond or any other bond known to those in the art asuseful for linking nucleotides to produce synthetic polynucleotides(see, for example, Tam et al., Nucl. Acids Res. 22:977-986, 1994); Eckerand Crooke, BioTechnology 13:351360, 1995, each of which is incorporatedherein by reference). The incorporation of non-naturally occurringnucleotide analogs or bonds linking the nucleotides or analogs can beparticularly useful where the polynucleotide is to be exposed to anenvironment that can contain a nucleolytic activity, including, forexample, a tissue culture medium, a cell or in a living subject, sincethe modified polynucleotides can be designed to be less (or, if desired,more) susceptible to degradation.

In general, the nucleotides comprising a polynucleotide are naturallyoccurring deoxyribonucleotides, such as adenine, cytosine, guanine orthymine linked to 2′-deoxyribose, or ribonucleotides such as adenine,cytosine, guanine or uracil linked to ribose. However, a polynucleotide(or oligonucleotide) also can contain nucleotide analogs, includingnon-naturally occurring synthetic nucleotides or modified naturallyoccurring nucleotides. Such nucleotide analogs are well known in the artand commercially available, as are polynucleotides containing suchnucleotide analogs (Lin et al., Nucl. Acids Res. 22:5220-5234, 1994;Jellinek et al., Biochemistry 34:11363-11372, 1995; Pagratis et al.,Nature Biotechnol. 15:68-73, 1997, each of which is incorporated hereinby reference).

A polynucleotide comprising naturally occurring nucleotides andphosphodiester bonds can be chemically synthesized or can be producedusing recombinant DNA methods, using an appropriate polynucleotide as atemplate. In comparison, a polynucleotide comprising nucleotide analogsor covalent bonds other than phosphodiester bonds generally will bechemically synthesized, although an enzyme such as T7 polymerase canincorporate certain types of nucleotide analogs into a polynucleotideand, therefore, can be used to produce such a polynucleotiderecombinantly from an appropriate template (Jellinek et al., supra,1995). As such, the polynucleotide can be prepared using a method suchas conventional phosphotriester and phosphodiester methods, including,for example, an automated method such as that usingdiethylphosphoramidites (see Beaucage et al., Tetrahedron Lett.,22:1859-1862, 1981), or a method whereby the oligonucleotides aresynthesized on a modified solid support (see U.S. Pat. No. 4,458,066).

An oligonucleotide of the invention, which can selectively hybridize toa target nucleic acid molecule and can be used as a reagent fordetecting methylation of a SOCS/CIS gene such as SOCS-1, is designed toselectively hybridize to a nucleotide sequence within about 2000nucleotides upstream (5′) or downstream (3′) of the target SOCS/CISgene, and generally within about 1000 nucleotides of the regioncomprising the CpG island that is to be examined for cytosinemethylation, usually within about 500 nucleotides of the site to beexamined. In addition, an oligonucleotide of the invention, or useful ina method of the invention, is at least about 12 nucleotides in length,generally at least about 14 or 15 nucleotides in length, and usually atleast about 18 to 20 nucleotides, such that it can selectively hybridizeto the target nucleic acid molecule. It will be recognized that thelength of the oligonucleotide will depend, in part, on the targetSOCS/CIS gene. For example, when the target SOCS/CIS gene is one of afamily of closely related genes having regions of substantial sequencesimilarity, a longer oligonucleotide can be used to assure selectivehybridization to the target oligonucleotide and minimal, if any,cross-hybridization to the related gene sequence(s).

Oligonucleotides of the invention are designed to be substantiallycomplementary to each strand of the genomic locus to be amplified and,where they are to be used for differentiating methylated fromunmethylated cytosine residues, will include the appropriate guanine orcytosine residues, as discussed above. Oligonucleotides of the inventionare exemplified herein by those having nucleotide sequences as set forthin SEQ ID NOS:6, 7, and 12 to 17, as well as those set forth in SEQ IDNOS:2 to 5, which can be used to differentiate a target sequencecontaining methylated cytosine residues of a CpG dinucleotide from onecontaining unmethylated cytosine residues.

Accordingly, the present invention provides an oligonucleotide selectedfrom any one of SEQ ID NOS:2 to 6 and 12 to 17, and further provides aplurality of such oligonucleotides, which includes at least two (e.g.,2, 3, 4, 5, or more) of the oligonucleotides set forth as SEQ ID NOS:2to 6 and 12 to 17. The present invention also provides an amplificationprimer pair, which comprises a forward primer and a reverse primer,particularly a primer pair that includes one of SEQ ID NOS:2 to 6 and 12to 17, which can be a forward primer or a reverse primer of the primerpair. Amplification primer pairs of the invention are exemplified hereinby those set forth as SEQ ID NOS:2 and 3; SEQ ID NOS: 4 and 5; SEQ IDNOS:6 and 7; SEQ ID NOS:12 and 13; SEQ ID NOS:14 and 15; and SEQ IDNOS:16 and 17. Furthermore, the present invention provides a pluralityof amplification primer pairs, which includes at least one of the primerpairs set forth as SEQ ID NO:2 and SEQ ID NO:3; SEQ ID NO: 4 and SEQ IDNO:5; SEQ ID NO:6 and SEQ ID NO:7; SEQ ID NO:12 and SEQ ID NO:13; SEQ IDNO:14 and SEQ ID NO:15; or SEQ ID NO:16 and SEQ ID NO:17, and a secondprimer pair, which can, but need not, be one of the above listed primerpairs.

Primer pairs of the invention further include methylation-specificprimer pairs, which only amplify a region of a CpG island of SOCS/CISgene containing methylated cytosine residues in CpG dinucleotides in theCpG island; and unmethylation-specific primer pairs, which amplify aregion of a CpG island of SOCS/CIS gene containing unmethylated cytosineresidues in CpG dinucleotide in the CpG island. A methylation-specificprimer pair of the invention is exemplified herein by the primer pairset forth in SEQ ID NOS:2 and 3, which can be used to specificallyamplify a methylated SOCS-1 gene sequence. An unmethylation-specificprimer pair of the invention is exemplified herein by the primer pairset forth as SEQ ID NOS:4 and 5, which can be used to specificallyamplify an unmethylated SOCS-1 gene sequence. As such, it will berecognized that a plurality of primer pairs of the invention can includeat least a first primer pair that is a methylation-specific primer pairsuch as the primer pair set forth as SEQ ID NOS:2 and 3, and a secondprimer pair that is an umethylation-specific primer pair such as theprimer pair set forth as SEQ ID NOS:4 and 5. In one embodiment, such aplurality of primer pairs includes the primer pairs set forth as SEQ IDNOS:2 and 3, and SEQ ID NOS:4 and 5. It should further be recognizedthat a plurality of primer pairs can include at least a first primerpair such as one of the primer pairs exemplified by SEQ ID NO:2 and SEQID NO:3; SEQ ID NO:4 and SEQ ID NO:5; SEQ ID NO:6 and SEQ ID NO:7; SEQID NO:12 and SEQ ID NO:13; SEQ ID NO:14 and SEQ ID NO:15; or SEQ IDNO:16 and SEQ ID NO:17; and a second primer pair that includesoligonucleotides that selectively hybridize to an amplification productgenerated using the first amplification primer pair, thus providingreagents useful for performing a nested amplification procedure.

The present invention also provides kits that contain one or morereagents useful for practicing a method of the invention. As such, a kitof the invention can contain, for example, one or more oligonucleotidesthat selectively hybridize to or near a CpG island of a SOCS/CIS gene,e.g., one or more of SEQ ID NOS:2 to 6 and 12 to 17. Such a kit can beuseful for preparing a probe that selectively hybridizes to theparticular SOCS/CIS gene sequence, in which case the kit can furthercontain, for example, a detectable label that can be linked to orincorporated into the probe, or a plurality of different detectablelabels such that, depending the needs of the user, can be selected for aparticular use, and, if desired, reagents for linking or incorporatingthe detectable label into the oligonucleotide. Alternatively, or inaddition, the kit can contain one or more reagents useful for performinga hybridization reaction such that selective hybridization conditionsreadily are attained; and/or can contain one or more standard nucleicacid molecules, for example, a standard target SOCS-1 nucleotidesequence that contains methylated cytosine residues corresponding theregion to which the oligonucleotide is designed to selectivelyhybridize, or a standard target SOCS-1 nucleotide sequence that containsunmethylated cytosine residues corresponding to the target sequence, ora combination thereof. Such standards provide several advantages,including, for example, allowing a confirmation that a reaction using atest cell, or extract thereof, functioned properly, or allowing forcomparisons among samples examined at different times or collected fromdifferent sources.

A kit containing one or more oligonucleotides of the invention such asone or more of SEQ ID NOS:2 to 6 and 12 to 17 also can be useful forperforming a primer extension reaction. As such, the kit can furtherinclude reagents for performing the selective hybridization reactionsuch that the oligonucleotide provides a substrate for the extensionreaction; and/or one or more reagents for performing the primerextension reaction, for example, dNTPs, one or more of which can bedetectably labeled or otherwise modified for conveniently linking adetectable label; and/or one or more standard target nucleic acidmolecules, as discussed above. Where a kit of the invention contains twoor more oligonucleotides (or primer pairs) such as those exemplifiedherein or otherwise useful for practicing the methods of the invention,the kit provides a convenient source of reagents from which the skilledartisan can select one or more oligonucleotides (or primer pairs), asdesired.

As such, a kit of the invention also can at least one amplificationprimer pair useful for detecting the presence or absence of methylatedcytosine residues in a CpG dinucleotide of a CpG island of a SOCS/CISgene. Amplification primer pairs that can be included in a kit of theinvention are exemplified by those set forth in SEQ ID NO:2 and SEQ IDNO:3; SEQ ID NO: 4 and SEQ ID NO:5; SEQ ID NO:6 and SEQ ID NO:7; SEQ IDNO:12 and SEQ ID NO:13; SEQ ID NO:14 and SEQ ID NO:15; and SEQ ID NO:16and SEQ ID NO:17. Such kits of the invention are further exemplified bya kit containing a plurality of primer pairs, including amethylation-specific primer pair and an unmethylation-specific primerpair, for example, a kit containing the primer pairs set forth as SEQ IDNOS:2 and 3, and SEQ ID NOS:4 and 5. Such a kit also can containreagents for providing conditions suitable for performing anamplification reaction, including, for example, dNTPs, one or aselection of polymerases, buffers, detectable labels, one or morestandard target nucleic acid molecules, a chemical reagent thatdifferentially modifies methylated as compared to unmethylated cytosineresidues, a methylation sensitive restriction endonuclease. and thelike.

As disclosed herein, methylation of CpG dinucleotides in a CpG island ofa SOCS/CIS gene such as SOCS-1 is associated with loss of regulation ofcell growth, including cancer. Accordingly, the invention providesmethods for selecting a therapeutic strategy for treating a cancerpatient by detecting methylation-silenced transcription of a SOCS/CISgene in cancer cells of the patient. Such a method can be performed byexamining a sample suspected of containing cancer cells from the patientfor decreased expression of a SOCS/CIS gene product due tomethylation-silenced transcription, whereby the detection of decreasedexpression of a SOCS/CIS gene product indicates selecting an agent thatrestores the SOCS/CIS gene product to the cancer cells as a component ofthe therapeutic strategy.

Upon determining that a cancer is associated with methylation-silencedtranscription of a SOCS/CIS gene, the agent selected for restoringSOCS/CIS gene product to the cell can be an agent that acts generally,for example, a demethylating agent, which restores transcriptionalactivity of the silenced SOCS/CIS gene, or an agent that modulates theactivity of an effector molecule downstream of the SOCS/CIS gene in asignal transduction pathway, for example, an agent such as AG490, whichinhibits JAK kinase activity. The agent to be selected also can be amore specific agent that is selected based on the particular SOCS/CISgene that is methylation-silenced. For example, where the cancer isassociated with methylation-silenced SOCS-1 gene expression, the agentcan be the SOCS-1 protein, or a polynucleotide encoding SOCS-1, such asthe polynucleotide set forth in SEQ ID NO:1, or a substantially similarpolynucleotide that has silent nucleotide changes that change a codon toa degenerate codon encoding the same amino acid.

Accordingly, the present invention also provides methods for reducing orinhibiting unregulated growth of a cell exhibiting methylation silencedtranscription of a SOCS/CIS gene, including methods that can beperformed in vitro or in vivo, and can be used for treating anindividual suffering from a disorder associated with such unregulatedgrowth, for example, a cancer patient, thereby providing a means toameliorate the severity of the disorder. The present invention alsoprovides compositions useful for practicing such methods and, therefore,further provides medicaments, which are useful for restoringmethylation-silenced transcription of a SOCS/CIS gene.

As a result of methylation-silenced transcription of a SOCS/CIS gene ina cell, the SOCS/CIS gene product is not present in the cell and,therefore, signal transduction pathways that include the SOCS/CIS geneproduct can exhibit aberrant regulation. For example,methylation-silenced transcription of the SOCS-1 gene results inconstitutively activated JAK2 kinase due to absence of the SOCS-1protein, which down-regulates JAK2 activity, and unregulated growth ofthe cell, which can be due, at least in part, to a decreased level ofapoptosis. Accordingly, the methods of the invention are based onproviding a cell that exhibits unregulated growth due tomethylation-silenced transcription of a SOCS/CIS gene with thepolypeptide encoded by the methylation-silenced SOCS/CIS gene, therebyrestoring regulated growth to the cell. As disclosed herein, thepolypeptide can be provided to the cell directly, can be expressed froman exogenous polynucleotide that is introduced into the cell and encodesthe polypeptide, or by restoring expression of the endogenousmethylation-silenced SOCS/CIS gene in the cell. By restoring theSOCS/CIS polypeptide to a cell exhibiting unregulated growth, orcharacteristics generally associated with unregulated growth, including,for example, the ability to grow in soft agar, a lack of contactinhibited growth, or refractoriness to programmed cell death, arealleviated.

A method of the invention can be practiced by restoring expression ofthe methylation-silenced SOCS/CIS gene in the cell. Such a method can bepracticed, for example, by contacting the cells with a demethylatingagent such as 5-azacytidine, which, when incorporated into the SOCS/CISgene during replication of a cell containing a methylation-silencedSOCS/CIS gene, results in progeny cells containing an unmethylatedSOCS/CIS gene. The cells contacted with the demethylating agent can becells in culture, wherein the demethylating agent is added to the cellculture medium in an amount sufficient to result in demethylation of theSOCS/CIS gene, without being toxic to the cells. The cells in culturecan be cells of an established cell line, or can be cells, which can bea mixed population of cells, that have been removed from a subject andare being contacted ex vivo, for example, to determine whether contactwith the particular demethylating agent can restore the SOCS/CIS geneexpression, and therefore, can be useful when administered to thesubject. Such ex vivo treatment of the cells also can be useful forrestoring SOCS/CIS gene expression, after which the cells, whichoptionally can be expanded in culture, can be administered back to thesubject. For example, bone marrow cells can be obtained by an aspirationor other method from an individual suffering from multiple myeloma or anacute leukemia, treated ex vivo with the demethylating agent, thenadministered back into the subject. Such a method, as well as any of themethods of treatment as disclosed herein, can further include treatmentsotherwise known in the art as useful for treating a subject having theparticular cancer, or that can be newly useful when used in combinationwith the present methods. For example, a patient with an acute leukemiacan be treated with a chemotherapeutic agent or with whole bodyradiotherapy to destroy leukemia cells in the body, then the ex vivotreated bone marrow cells, which comprises cells having restoredSOCS/CIS gene expression, can be administered back into the patient.

Cells exhibiting methylation-silenced SOCS/CIS gene expression also canbe contacted with the demethylating agent in vivo by administering theagent to a subject. Where convenient, the demethylating agent can beadministered using, for example, a catheterization procedure, at or nearthe site of the cells exhibiting unregulated growth in the subject, orinto a blood vessel in which the blood is flowing to the site of thecells. Similarly, where an organ to be treated can be isolated by ashunt procedure, the agent can be administered via the shunt, thussubstantially providing the agent to the site containing the cells. Theagent also can be administered systemically or via other routes asdisclosed herein or otherwise known in the art.

A method of providing a SOCS/CIS polypeptide to a cell also can beperformed by introducing a polynucleotide encoding the SOCS/CISpolypeptide into the cell, whereby the SOCS/CIS polypeptide is expressedfrom the polynucleotide. As such, the present invention provides methodsof gene therapy, which can be practiced in vivo or ex vivo. For example,where the cell is characterized by methylation-silenced transcription ofthe SOCS-1 gene, a polynucleotide comprising SEQ ID NO:1 can becontacted with the target cell.

The polynucleotide encoding the SOCS/CIS polypeptide can include, inaddition to SOCS/CIS polypeptide coding sequence, operatively linkedtranscriptional regulatory elements, translational regulatory elements,and the like, and can be in the form of a naked DNA molecule, or can beformulated in a matrix that facilitates entry of the polynucleotide intothe particular cell, for example, a liposome, in which case thepolynucleotide contains the required operatively linked regulatoryelements. As used herein, the term “operatively linked” refers to two ormore molecules are positioned with respect to each other such that theyact as a single unit and effect a function attributable to one or bothmolecules or a combination thereof. For example, a polynucleotideencoding a SOCS-1 polypeptide can be operatively linked to a second (ormore) coding sequence, such that a chimeric polypeptide can be expressedfrom the operatively linked coding sequences. The chimeric polypeptidecan be a fusion protein, in which the two (or more) encoded polypeptidesare translated into a single polypeptide, i.e., are covalently boundthrough a peptide bond; or can be translated as two discrete peptidesthat, upon translation, can operatively associate with each other toform a stable complex. Similarly, a polynucleotide sequence encoding aSOCS/CIS polypeptide can be operatively linked to a regulatory element,in which case the regulatory element confers its regulatory effect onthe polynucleotide similarly to the way in which the regulatory elementwould effect a polynucleotide sequence with which it normally isassociated with in a cell.

A fusion protein generally demonstrates some or all of thecharacteristics of each of its polypeptide components, and, therefore,can be useful for restoring SOSC/CIS gene expression in the cell and canfurther provide additional advantages. For example, the fusion proteincan include a SOCS-1 polypeptide operatively linked to a cellcompartment localization domain such that expression of the fusionprotein in a cell or loading of the cell with fusion protein allowstranslocation of the SOCS-1 polypeptide (or other SOCS/CIS polypeptide)to the intracellular compartment such as the nucleus, in which iteffects its activity. Cell compartmentalization domains, for example,are well known and include a plasma membrane localization domain, anuclear localization signal, a mitochondrial membrane localizationsignal, an endoplasmic reticulum localization signal, and the like, aswell as signal peptides, which can direct secretion of a polypeptidefrom a cell (see, for example, Hancock et al., EMBO J. 10:4033-4039,1991; Buss et al., Mol. Cell. Biol. 8:3960-3963, 1988; U.S. Pat. No.5,776,689 each of which is incorporated herein by reference). The fusionprotein also can comprise a SOCS/CIS polypeptide operatively linked to apeptide that acts as a ligand for a receptor, a peptide useful as a tagfor identifying a cell containing the SOCS/CIS polypeptide, or forisolating the fusion protein, or any other peptide or polypeptide ofinterest, providing the fusion protein has the SOCS/CIS protein activityfor which it is being expressed, e.g., to reduce or inhibit constitutiveJAK activity. Peptide tags such as a polyhistidine tag peptide, e.g.,His-6, which can be detected using a divalent cation such as nickel ion,cobalt ion, or the like; a FLAG epitope, which can be detected using ananti-FLAG antibody (see, for example, Hopp et al., BioTechnology 6:1204(1988); U.S. Pat. No. 5,011,912, each of which is incorporated herein byreference); a c-myc epitope, which can be detected using an antibodyspecific for the epitope; biotin, which can be detected usingstreptavidin or avidin; and glutathione S-transferase, which can bedetected using glutathione, are well known in the art, and provide ameans of detecting the presence of a SOCS/CIS polypeptide operativelylinked thereto. Such tags provide the additional advantage that they canfacilitate isolation of the operatively linked SOCS/CIS polypeptide, forexample, where it is desired to obtain the SOCS/CIS polypeptide in asubstantially purified form, such a polypeptide also being useful forpracticing methods of the invention.

A polynucleotide encoding a SOCS/CIS gene product can be used alone, orcan be contained in a vector, which can facilitate manipulation of thepolynucleotide, including introduction of the polynucleotide into atarget cell. The vector can be a cloning vector, which is useful formaintaining the polynucleotide, or can be an expression vector, whichcontains, in addition to the polynucleotide, regulatory elements usefulfor expressing the polynucleotide and encoded SOCS/CIS polypeptide in aparticular cell. An expression vector can contain the expressionelements necessary to achieve, for example, sustained transcription ofthe encoding polynucleotide, or the regulatory elements can beoperatively linked to the polynucleotide prior to its being cloned intothe vector.

An expression vector (or the polynucleotide encoding the SOCS/CISpolypeptide) generally contains or encodes a promoter sequence, whichcan provide constitutive or, if desired, inducible or tissue specific ordevelopmental stage specific expression of the encoding polynucleotide,a poly-A recognition sequence, and a ribosome recognition site orinternal ribosome entry site, or other regulatory elements such as anenhancer, which can be tissue specific. The vector also can containelements required for replication in a prokaryotic or eukaryotic hostsystem or both, as desired. Such vectors, which include plasmid vectorsand viral vectors such as bacteriophage, baculovirus, retrovirus,lentivirus, adenovirus, vaccinia virus, semliki forest virus andadeno-associated virus vectors, are well known and can be purchased froma commercial source (Promega, Madison Wis.; Stratagene, La Jolla Calif.;GIBCO/BRL, Gaithersburg Md.) or can be constructed by one skilled in theart (see, for example, Meth. Enzymol., Vol. 185, Goeddel, ed. (AcademicPress, Inc., 1990); Jolly, Canc. Gene Ther. 1:51-64, 1994; Flotte, J.Bioenerg. Biomemb. 25:37-42, 1993; Kirshenbaum et al., J. Clin. Invest.92:381-387, 1993; each of which is incorporated herein by reference).

A tetracycline (tet) inducible promoter can be particularly useful fordriving expression of a polynucleotide encoding a SOCS/CIS polypeptide.Upon administration of tetracycline, or a tetracycline analog, to asubject containing a polynucleotide operatively linked to a tetinducible promoter, expression of the encoded SOCS/CIS polypeptide isinduced. The polynucleotide also can be operatively linked to tissuespecific regulatory element, for example, a liver cell specificregulatory element such as an I-fetoprotein promoter (Kanai et al.,Cancer Res. 57:461-465, 1997; He et al., J. Exp. Clin. Cancer Res.19:183-187, 2000) or an albumin promoter (Power et al., Biochem.Biophys. Res. Comm. 203:1447-1456, 1994; Kuriyama et al., Int. J. Cancer71:470-475, 1997); a muscle cell specific regulatory element such as amyoglobin promoter (Devlin et al., J. Biol. Chem. 264:13896-13901, 1989;Yan et al., J. Biol. Chem. 276:17361-17366, 2001); a prostate cellspecific regulatory element such as the PSA promoter (Schuur et al., J.Biol. Chem. 271:7043-7051, 1996; Latham et al., Cancer Res. 60:334-341,2000); a pancreatic cell specific regulatory element such as theelastase promoter (Ornitz et al., Nature 313:600-602, 1985; Swift etal., Genes Devel. 3:687-696, 1989); a leukocyte specific regulatoryelement such as the leukosialin (CD43) promoter (Shelley et al.,Biochem. J. 270:569-576, 1990; Kudo and Fukuda, J. Biol. Chem.270:13298-13302, 1995); or the like, such that expression of thepolypeptide is restricted to particular cell in an individual, or toparticular cells in a mixed population of cells in culture, for example,an organ culture. Regulatory elements, including tissue specificregulatory elements, many of which are commercially available, are wellknown in the art (see, for example, InvivoGen; San Diego Calif.).

Viral expression vectors can be particularly useful for introducing apolynucleotide into a cell, particularly a cell in a subject. Viralvectors provide the advantage that they can infect host cells withrelatively high efficiency and can infect specific cell types. Forexample, a polynucleotide encoding a SOCS/CIS polypeptide can be clonedinto a baculovirus vector, which then can be used to infect an insecthost cell, thereby providing a means to produce large amounts of theencoded polypeptide. The viral vector also can be derived from a virusthat infects cells of an organism of interest, for example, vertebratehost cells such as mammalian, avian or piscine host cells. Viral vectorscan be particularly useful for introducing a polynucleotide useful inperforming a method of the invention into a target cell. Viral vectorshave been developed for use in particular host systems, particularlymammalian systems and include, for example, retroviral vectors, otherlentivirus vectors such as those based on the human immunodeficiencyvirus (HIV), adenovirus vectors, adeno-associated virus vectors,herpesvirus vectors, hepatitis virus vectors, vaccinia virus vectors,and the like (see Miller and Rosman, BioTechniques 7:980-990, 1992;Anderson et al., Nature 392:25-30 Suppl., 1998; Verma and Somia, Nature389:239-242, 1997; Wilson, New Engl. J. Med. 334:1185-1187 (1996), eachof which is incorporated herein by reference).

A polynucleotide, which can be contained in a vector, can be introducedinto a cell by any of a variety of methods known in the art (Sambrook etal., supra, 1989; Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Baltimore, Md. (1987, and supplementsthrough 1995), each of which is incorporated herein by reference). Suchmethods include, for example, transfection, lipofection, microinjection,electroporation and, with viral vectors, infection; and can include theuse of liposomes, microemulsions or the like, which can facilitateintroduction of the polynucleotide into the cell and can protect thepolynucleotide from degradation prior to its introduction into the cell.The selection of a particular method will depend, for example, on thecell into which the polynucleotide is to be introduced, as well aswhether the cell is isolated in culture, or is in a tissue or organ inculture or in situ.

Introduction of a polynucleotide into a cell by infection with a viralvector is particularly advantageous in that it can efficiently introducethe nucleic acid molecule into a cell ex vivo or in vivo (see, forexample, U.S. Pat. No. 5,399,346, which is incorporated herein byreference). Moreover, viruses are very specialized and can be selectedas vectors based on an ability to infect and propagate in one or a fewspecific cell types. Thus, their natural specificity can be used totarget the nucleic acid molecule contained in the vector to specificcell types. As such, a vector based on an HIV can be used to infect Tcells, a vector based on an adenovirus can be used, for example, toinfect respiratory epithelial cells, a vector based on a herpesvirus canbe used to infect neuronal cells, and the like. Other vectors, such asadeno-associated viruses can have greater host cell range and,therefore, can be used to infect various cell types, although viral ornon-viral vectors also can be modified with specific receptors orligands to alter target specificity through receptor mediated events. Apolynucleotide of the invention, or a vector containing thepolynucleotide can be contained in a cell, for example, a host cell,which allows propagation of a vector containing the polynucleotide, or ahelper cell, which allows packaging of a viral vector containing thepolynucleotide. The polynucleotide can be transiently contained in thecell, or can be stably maintained due, for example, to integration intothe cell genome.

A method of the invention also can be practiced by directly providingSOCS/CIS polypeptide to a cell exhibiting unregulated growth. TheSOCS/CIS polypeptide can be produced and isolated, and formulated asdesired, using methods as disclosed herein. The polypeptide can becontacted with the cell in vitro under conditions that result insufficient permeability of the cell such that the polypeptide can crossthe cell membrane, or can be microinjected into the cells. Where theSOCS/CIS polypeptide is contacted with a cell in situ in an organism, itcan comprise a fusion protein, which includes a peptide or polypeptidecomponent that facilitates transport across the cell membrane, forexample, a human immunodeficiency virus (HIV) TAT protein transductiondomain, and can further comprise a nuclear localization domainoperatively linked thereto. Alternatively, or in addition, thepolypeptide can be formulated in a matrix that facilitates entry of thepolypeptide into a cell.

In one embodiment, the invention provides a method of treating a subjectsuffering from hepatocellular carcinoma (HCC) or multiple myeloma (MM),wherein cells associated with the HCC or MM exhibit methylation silencedSOCS-1 gene expression, the method comprising administering an amount ofAG490 to the subject sufficient to induce apoptosis of the cellsassociated with the cancer. In another embodiment, the inventionprovides a method for treating a cancer patient, wherein cancer cells inthe patient exhibit methylation silenced SOCS-1 gene expression, byproviding SOCS-1 polypeptide (SEQ ID NO:20) to the cancer cells, therebyinducing apoptosis of the cells. SOCS-1 polypeptide can be provided tothe cancer cells by contacting the cells with a demethylating agent, forexample, by administering the demethylating agent to the subject in anamount sufficient to restore SOCS-1 gene expression in the cancer cells,or by introducing a polynucleotide encoding SOCS-1, for example, thepolynucleotide set forth as SEQ ID NO:1 or a polynucleotide encoding SEQID NO:20, into the cancer cells under conditions sufficient forexpression of the encoded SOCS-1 polypeptide in the cancer cells. Wherethe polynucleotide is administered to a subject, the polynucleotide canbe contained in a vector, particularly a vector derived from a virusthat preferentially infects the cells from the cancer cells arose, forexample, a vector derived from or containing components of a hepatitisvirus where the cancer cells are hepatocellular carcinoma cells, or avector derived from or containing components of HIV where the cancercells are T cell leukemia cells. The polynucleotide, which can becontained in a vector, also can be formulated with a matrix such as aliposome, which can be further modified to contain a receptor (orligand) on its surface, wherein the receptor (or ligand) canspecifically bind a cognate ligand (or receptor) expressed by the cancercells, for example, the liposome can contain on its surface antibodiessuch as anti-idiotype antibodies that specifically binds with antibodiesexpressed by plasma cells associated with a multiple myeloma, or cancontain a ligand that specifically with an apolipoprotein expressed byhepatocytes.

For administration to a living subject, an agent such as a demethylatingagent, AG490, a polynucleotide encoding a SOCS/CIS gene, or a SOCS/CISpolypeptide useful for practicing a therapeutic method of the inventiongenerally is formulated in a composition suitable for administration tothe subject. Thus, the invention provides compositions containing anagent that is useful for restoring regulated growth to a cell exhibitingunregulated growth due to methylation-silenced transcription of aSOCS/CIS gene. As such, the agents are useful as medicaments fortreating a subject suffering from a pathological condition associatedwith such unregulated growth.

Such compositions generally include a carrier that can is acceptable forformulating and administering the agent to a subject. Such acceptablecarriers are well known in the art and include, for example, aqueoussolutions such as water or physiologically buffered saline or othersolvents or vehicles such as glycols, glycerol, oils such as olive oilor injectable organic esters. An acceptable carrier can containphysiologically acceptable compounds that act, for example, to stabilizeor to increase the absorption of the conjugate. Such physiologicallyacceptable compounds include, for example, carbohydrates, such asglucose, sucrose or dextrans, antioxidants, such as ascorbic acid orglutathione, chelating agents, low molecular weight proteins or otherstabilizers or excipients. One skilled in the art would know that thechoice of an acceptable carrier, including a physiologically acceptablecompound, depends, for example, on the physico-chemical characteristicsof the therapeutic agent and on the route of administration of thecomposition, which can be, for example, orally or parenterally such asintravenously, and by injection, intubation, or other such method knownin the art. The pharmaceutical composition also can contain a secondreagent such as a diagnostic reagent, nutritional substance, toxin, ortherapeutic agent, for example, a cancer chemotherapeutic agent.

The agent can be incorporated within an encapsulating material such asinto an oil-in-water emulsion, a microemulsion, micelle, mixed micelle,liposome, microsphere or other polymer matrix (see, for example,Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton, Fla.1984); Fraley, et al., Trends Biochem. Sci., 6:77 (1981), each of whichis incorporated herein by reference). Liposomes, for example, whichconsist of phospholipids or other lipids, are nontoxic, physiologicallyacceptable and metabolizable carriers that are relatively simple to makeand administer. “Stealth” liposomes (see, for example, U.S. Pat. Nos.5,882,679; 5,395,619; and 5,225,212, each of which is incorporatedherein by reference) are an example of such encapsulating materialsparticularly useful for preparing a composition useful in a method ofthe invention, and other “masked” liposomes similarly can be used, suchliposomes extending the time that the therapeutic agent remain in thecirculation. Cationic liposomes, for example, also can be modified withspecific receptors or ligands (Morishita et al., J. Clin. Invest.,91:2580-2585 (1993), which is incorporated herein by reference). Inaddition, a polynucleotide agent can be introduced into a cell using,for example, adenovirus-polylysine DNA complexes (see, for example,Michael et al., J. Biol. Chem. 268:6866-6869 (1993), which isincorporated herein by reference).

The route of administration of the composition containing thetherapeutic agent will depend, in part, on the chemical structure of themolecule. Polypeptides and polynucleotides, for example, are notparticularly useful when administered orally because they can bedegraded in the digestive tract. However, methods for chemicallymodifying polypeptides, for example, to render them less susceptible todegradation by endogenous proteases or more absorbable through thealimentary tract are disclosed herein or otherwise known in the art(see, for example, Blondelle et al., supra, 1995; Ecker and Crook,supra, 1995). In addition, a polypeptide agent can be prepared usingD-amino acids, or can contain one or more domains based onpeptidomimetics, which are organic molecules that mimic the structure ofa domain; or based on a peptoid such as a vinylogous peptoid.

A composition as disclosed herein can be administered to an individualby various routes including, for example, orally or parenterally, suchas intravenously, intramuscularly, subcutaneously, intraorbitally,intracapsularly, intraperitoneally, intrarectally, intracisternally orby passive or facilitated absorption through the skin using, forexample, a skin patch or transdermal iontophoresis, respectively.Furthermore, the composition can be administered by injection,intubation, orally or topically, the latter of which can be passive, forexample, by direct application of an ointment, or active, for example,using a nasal spray or inhalant, in which case one component of thecomposition is an appropriate propellant. A pharmaceutical compositionalso can be administered to the site of a pathologic condition, forexample, intravenously or intra-arterially into a blood vessel supplyinga tumor.

The total amount of an agent to be administered in practicing a methodof the invention can be administered to a subject as a single dose,either as a bolus or by infusion over a relatively short period of time,or can be administered using a fractionated treatment protocol, in whichmultiple doses are administered over a prolonged period of time. Oneskilled in the art would know that the amount of the composition totreat a pathologic condition in a subject depends on many factorsincluding the age and general health of the subject as well as the routeof administration and the number of treatments to be administered. Inview of these factors, the skilled artisan would adjust the particulardose as necessary. In general, the formulation of the composition andthe routes and frequency of administration are determined, initially,using Phase I and Phase II clinical trials.

The composition can be formulated for oral formulation, such as atablet, or a solution or suspension form; or can comprise an admixturewith an organic or inorganic carrier or excipient suitable for enteralor parenteral applications, and can be compounded, for example, with theusual non-toxic, pharmaceutically acceptable carriers for tablets,pellets, capsules, suppositories, solutions, emulsions, suspensions, orother form suitable for use. The carriers, in addition to thosedisclosed above, can include glucose, lactose, mannose, gum acacia,gelatin, mannitol, starch paste, magnesium trisilicate, talc, cornstarch, keratin, colloidal silica, potato starch, urea, medium chainlength triglycerides, dextrans, and other carriers suitable for use inmanufacturing preparations, in solid, semisolid, or liquid form. Inaddition auxiliary, stabilizing, thickening or coloring agents andperfumes can be used, for example a stabilizing dry agent such astriulose (see, for example, U.S. Pat. No. 5,314,695).

The following examples are intended to illustrate, but not limit, theinvention.

EXAMPLE 1 Methylation of SOCS-1 is Associated with HepatocellularCarcinoma

This example demonstrates that hypermethylation of the SOCS-1 gene andconsequent decreased expression of SOCS-1 correlate with unregulatedgrowth of hepatocellular carcinoma (HCC) cells, and that agents thatcircumvent the decreased SOCS-1 expression induce apoptosis of the HCCcells.

Methods

Cell Lines and Tissue Samples

Human hepatocellular carcinoma cell (HCC) lines SNU-182, SNU-423,SNU-387, SNU-398, SNU-449, SNU-475, and PLC/PRF/5 were obtained fromAmerican Type Culture Collection. GM06061C was obtained from NIGMSrepository. HuH-1, HuH-4, HuH-7 and Hep3B were obtained from JapaneseCulture Collection. The cells were grown in DMEM or RPMI 1640supplemented with 10% fetal bovine serum (FBS) for isolation of DNA andRNA. Primary hepatocarcinoma samples and adjacent normal liver tissueare described by Rashid et al. (Brit. J. Cancer 80:59-66, 1999).

Northern Blot Analysis

Poly(A) RNA of HCC cell lines was prepared using the QuickPrep MICROmRNA Purification Kit (Amersham Pharmacia Biotech). Normal liver poly(A)RNA was purchased from Clontech (Palo Alto Calif.). The 3′ sequence ofSOCS-1 (nucleotides 679 to 1031 of GenBank Acc. No. U88326; SEQ ID NO:1;see, also, Starr et al., supra, 1997) was used as a probe. Northern blotanalysis was performed as described by Yoshikawa et al. (Genomics49:237-246, 1998); full length SOCS-1 mRNA migrates with a size of about1.3 kb. As a control, the same membrane was re-probed with a GAPD(G3PDH) probe (Clontech).

Methylation Specific PCR

Genomic DNA was extracted using a standard method, and bisulfitemodification of genomic DNA was performed as by Herman et al. (Proc.Natl. Acad. Sci. USA 93:9821-9826, 1996). Methylation status of SOCS-1was analyzed by methylation-specific PCR (MSP), which distinguishesunmethylated alleles from methylated alleles in a gene on the basis ofsequence changes induced by sodium bisulfite treatment of DNA; bisulfitetreatment converts all unmethylated, but not methylated, cytosineresidues to uracil. The DNA region of interest then was amplified withprimer pairs specific for methylated versus unmethylated DNA.

The bisulfite treated DNA was amplified either with amethylation-specific primer set or a unmethylation-specific primer setat 35 cycles for 95° C., 30 sec; 60° C., 30 sec; and 72° C., 30 sec.

The methylation specific primer sequences were as follows: forwardprimer-5′-TTCGCGTGTATTTTTAGGTCGGTC-3′ (SEQ ID NO:2), and reverseprimer-5′-CGACACAACTCCTACAACGACCG-3′ (SEQ ID NO:3). These primers weredesigned from nucleotides 400 to 423 (forward primer) and nucleotides537 to 559 (reverse primer) of the SOCS-1 sequence (SEQ ID NO:1).

The unmethylation specific primer sequences were as follows:

(SEQ ID NO: 4) forward 5′-TTATGAGTATTTGTGTGTATTTTTAGGTTGGTT-3′, primerand (SEQ ID NO: 5) reverse 5′-CACTAACAACACAACTCCTACAACAACCA-3′. primerThese primers were designed from nucleotides 391 to 423 (forward primer)and nucleotides 537 to 565 (reverse primer) of SEQ ID NO:1.

Amplification of methylated SOCS-1 using SEQ ID NOS:2 and 3 generates a160 bp DNA product, whereas amplification of unmethylated SOCS-1 usingSEQ ID NOS:4 and 5 generates a 175 bp product.

Sequencing Analysis

Bisulfite sequencing analysis was performed for 5 HCC cell lines and 2non-tumor liver samples. Bisulfite treated DNA was amplified using theprimer set: 5′-TGTAGGATGGTAGTATATAATTAGGTGGT-3′ (SEQ ID NO:6), and5′-TAATACTCCAACAACTCTAAAAAACAATC-3′ (SEQ ID NO:7), which was designed toamplify nucleotides 18 to 484 in SEQ ID NO:1. The PCR product was clonedinto the pCR2.1-TOPO vector (Invitrogen Corp.; Carlsbad Calif.), and 4to 8 randomly picked clones were sequenced using ABI PRISM Big DyeTerminator Cycle sequencing kit (Applied Biosystems, Inc.; Foster CityCalif.) according to the protocol supplied by the manufacturer.

Preparations of the Matrix and PCR Analysis of the Matrix Associated DNA

Matrix was isolated as described by Kramer et al. (supra, 1998).Briefly, 2×10⁶ cells were washed with phosphate buffered saline (PBS).Nuclei were isolated by treatment with a buffer containing 10 mM Tris(pH 7.7), 100 mM NaCl, 0.3 M sucrose, 3 mM MgCl₂ and 0.5% TRITON-X100detergent, and collected by centrifugation at 500×g for 5 min. Thenuclei were treated with 2M NaCl, 10 mM Tris pH 7.7 and 10 mM EDTA, andplaced on ice for 15 min.

The resulting halo structure was recovered by centrifugation, and washed3 times with a low salt buffer containing 10 mM Tris (pH 7.5), 10 mMMgCl₂ and 1 mM DTT. The halo was resuspended with the low salt buffer,and digested with 300 units of Sac I for 4 hr at 37° C. Completedigestion was assessed using a plasmid DNA as an internal marker.Digestion was terminated by addition of 0.5 M EDTA to a finalconcentration of 25 mM, then NaCl was added to a final concentration of2M, and the sample was incubated at 37° C. for 15 min. The pellet andsupernatant were fractionated by centrifugation at 12,000×g for 10 min.After washing the pellet, the matrix-attached DNA and matrix unattachedDNA were extracted by proteinase K treatment, followed byphenol/chloroform extraction from the pellet and supernatant fraction,respectively.

Using 50 ng of DNA, PCR was carried out with 30 cycles at 94° C., 40sec; 60° C., 1 min; and 72° C., 40 sec. The primers used for this methodwere as follows:

-   5′-AACACCCCAGCCATGTACG-3′ (SEQ ID NO:8), and    5′-ATGTCACGCACGATTTCCC-3′ (SEQ ID NO:9), for ACTB,-   5′-CAGCCCAGAGGAGCCTAAAG-3′ (SEQ ID NO:10), and    5′-TCCAGTTCAGGGTGCCATAC-3′ (SEQ ID NO:11) for human protamine 1    (PRM1; amplifies nucleotides 9112 to 9304 of GenBank Acc. No.    U15422); and-   5′-TTCTCTCACCCCCTCACGC-3′(SEQ ID NO:12), and    5′-GCTGGGCACTTGGTTACTGG-3′ (SEQ ID NO:13) for SOCS-1 (amplifies    nucleotides 38526 to 38834 of GenBank Acc. No. U15422, which is    incorporated herein by reference; see, also, Nelson and Krawetz, J.    Biol. Chem. 269:31067-31073, 1994; Kramer and Krawetz, BioTechniques    22:826-828, 1997, each of which is incorporated herein by    reference).    Immunoprecipitation and Western Blot Analysis

Cells at approximately 70% confluence were harvested and lysed on ice ina buffer containing 20 mM Tris (pH 8.0), 1% NONIDET P-40 detergent, 0.1%SDS, 150 mM NaCl, 50 mM NaF, 1 mM Na₃VO₄, 10 μg/ml leupeptin, 10 μg/mlaprotinin, 1 μg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride.Following incubation for 30 min on ice, the lysates were cleared ofdebris by centrifugation at 15,000×g for 30 min. Eight hundred μg ofprotein lysates were incubated with 5 μg anti-JAK2 antibody (UpstateBiotechnology; Waltham Mass.) for 1 hr on ice. Thirty μl protein A+G(Oncogene Research Products; Boston Mass.) was added to the lysates andincubated for 1 hr at 4° C. with rotation.

Immune complexes were recovered by centrifugation, washed 5 times withthe lysis buffer, then boiled for 5 min in SDS-PAGE sample buffer. Thesamples were resolved by SDS-PAGE, and electroblotted onto anitrocellulose membrane. The blot was blocked with 5% skim milk in PBSfor 1 hr at room temperature (RT), then incubated with 1 μg/ml ofanti-phosphotyrosine antibody (4G10; Upstate biotechnology) over nightat 4° C.

After several washes, a 1:5000 dilution of anti-mouse horseradishperoxidase-conjugated secondary antibody (Amersham Pharmacia Biotech)was added and incubated for 1 hr at RT. After several washes, theimmunoreactive bands were visualized by an enhanced chemiluminescencesubstrate (Amersham Pharmacia Biotech) and exposed to HYPERFILM film(Amersham Pharmacia Biotech). The blot was stripped with a buffercomposed of 62.5 mM Tris (pH 6.8), 2% SDS and 100 mM θ-mercaptoethanolat 50° C. for 30 min, washed, blocked with 5% milk in PBS, thenre-probed with the anti-JAK2 antibody (Upstate Biotechnology).

To analyze inhibition of STAT3 by AG490, cells were treated with 25 μMAG490 for 3 hr or 24 hr. Eighty μg total protein samples from drugtreated and untreated cells were separated by SDS-PAGE, and analyzed bywestern blot analysis. Anti-phospho-STAT3 antibody (New England Biolabs)was used to detect tyrosine phosphorylation of STAT3 according to theconditions recommended by the manufacturer. After removing the antibody,the blot was analyzed with anti-STAT3 antibody (Santa CruzBiotechnology).

Construction of Human SOCS-1 Expression Vector

A full length SOCS-1 cDNA was isolated from human embryonic liver RNA(Clontech) by PCR using the following primer set:

-   5′-CCCCTTCTGTAGGATGGTAG-3′ (SEQ ID NO:14), and    5′-CATCCCAGTTAATGCTGCGT-3′ (SEQ ID NO:15). The PCR product was    cloned into the pCR3.1 vector (Invitrogen). The full length SOCS-1    cDNA was excised from the recombinant vector with Eco RI, and    ligated with Eco RI-digested pcDNA3.1/H is C vector (Invitrogen). A    clone, pcDNA-SOCS-1, contained an in-frame ligation with the sense    orientation and a correct sequence when compared to GenBank Acc. No.    U88326 (SEQ ID NO:1).    Transfection Experiments and Colony Formation Assay with AG490

For colony formation in monolayer culture analysis, cells were plated at30×10⁴ cells per well in 6 well plates, and transfected with 5 μg ofeither pcDNA3.1-SOCS-1 or the backbone pcDNA3.1 vector (control) usingthe LipofectAMINE PLUS transfection reagent (Invitrogen Corp.) accordingto the protocol provided by the manufacturer. The cells were strippedand plated on 100 mm tissue culture at 48 hours post-transfection. Thecells were selected with G418 antibiotic at a concentration of 500μg/ml, and the colonies were counted 4 weeks after transfection.

For colony formation in soft agar analysis, cells were transfected asabove, and suspended in RPMI 1640 containing 0.35% agar, 10% fetalbovine serum and 500 μg/ml G418, and layered on RPMI 1640 containing0.5% agar, 10% fetal bovine serum and 500 μg/ml G418 in 100 mm tissueculture dishes at 48 hours post-transfection. An additional 0.35% freshagar culture medium with 500 μg/ml G418 was layered every 5 days. Colonyformation was assessed at 4 weeks post-transfection.

For AG490 treatment, 1×10⁴ cells were plated in 100 mm tissue culturedishes, and grown in complete medium in the absence or presence of 5 μMAG490 for 3 weeks. In some experiments, cells were treated with 25 μMAG490 for 3 hr or 24 hr.

Immunofluorescence Analysis

Approximately 3000 cells were seeded in an eight well chamber slide, andtransfected with 200 ng pcDNA3.1-SOCS-1 using the LipofectAMINE PLUStransfection reagent. At two days post-transfection, a TUNEL reactionwas performed using the In situ Cell Death Detection Kit, Fluoresceinkit (Roche Molecular Biochemicals) according to the protocol provided bythe manufacturer. Subsequently, the cells were blocked for 1 hr with 3%BSA at RT, then incubated with a 1:200 dilution of anti-Xpress™ antibody(Invitrogen Corp.) for 1 hr at 37° C. After several washes, the cellswere incubated with a 1:200 dilution of Texas red anti-mouse secondaryantibody (Vector Laboratories; Burlingame Calif.). The cells werewashed, and mounted in a medium (Vector Laboratories) containing 0.5μg/ml DAPI, then viewed under a fluorescence microscopy at 60×magnification. Exogenously expressed SOCS-1 protein, fragmented DNA, andnuclei were identified under appropriate filters.

Results

Methylation in SOCS-1 CpG Island Correlates with Silencing of GeneExpression

Methylation of the SOCS-1 CpG island was analyzed in eleven differentHCC cell lines. Methylation specific PCR (MSP) analysis revealedaberrant DNA methylation in 7 of the 11 HCC cell lines using a primerset that was designed in exon 1 and lies within the SOCS-1 CpG island.In contrast, one lymphoblast cell line and two non-tumor liver samples,including one from a cirrhotic liver and another from a patient withchronic hepatitis, did not show methylation in this region (seeYoshikawa et al., supra, 2001).

To examine DNA methylation patterns in the SOCS-1 CpG island in moredetail, the HCC cell lines were examined using bisulfite sequencing. Atotal of 58 CpG sites in exon 1 were examined in the 467 base pairgenomic sequence. HCC cell lines (Hep3B, PLC/PRF/5, and SNU-387), whichwere methylated when examined by MSP, also were densely methylated whenexamined by bisulfite sequencing. In addition, the HuH-1 cell linedemonstrated a regional and clustering methylation; MSP did not detectmethylation of this cell line because the CpG sites involved were not inthe same region as that examined by MSP. In contrast, no significantmethylation was detected in the two non-tumor liver samples or in theSNU-182 cells (see FIG. 1 in Yoshikawa et al., supra, 2001).

SOCS-1 expression in the HCC cell lines also was examined by northernblot analysis. Cell lines with fully unmethylated DNA (HuH-4, HuH-7 andSNU-182) and normal liver demonstrated substantial SOCS-1 expression. Incontrast, the methylated cell lines, HuH-1, Hep3B, SNU-387, SNU-449,PLC/PRF/5 and SNU-398 (as detected by MSP or bisulfite sequencing) didnot express SOCS-1. Interestingly, partially methylated cell lines(SNU-423 and SNU-475), in which half of the DNA was unmethylated, alsoexpressed SOCS-1 (see Table 1). As a control, GAPD probing of the samefilter demonstrated that the loading of the RNA was similar. Theseresults indicate the expression status of SOCS-1 correlated with themethylation status of the SOCS-1 CpG island (Table 1); i.e.,hypermethylation correlated with a lack of SOCS-1 gene expression.

TABLE 1 DNA methylation and RNA expression of SOCS-1 RNA MSP BisulfiteCell line expression ME UN sequencing HuH-1 − − + + Hep3B − + − +SNU-387 − + − + SNU-449 − + − PLC/PRF/5 − + − + SNU-398 − + −SNU-423 + + + SNU-475 + + + HuH-4 + − + HuH-7 + − + SNU-182 + − + −normal + A ‘−’ represents no expression and a ‘+’ represents significantRNA expression in northern-blot analysis. ‘ME’ and ‘UN’ indicate resultsfrom MSP analysis for methylation and unmethylation reactions,respectively, with ‘−’ indicating no product, whereas ‘+’ indicates thepresence of product. A ‘+’ in bisulfite sequencing section representsmethylation, and a ‘−’ represents unmethylation.Methylation Silencing is Associated with Alteration of the MatrixAssociation at the SOCS-1 CpG Island

Specific regions of chromosomal DNA are believed to attach to thenuclear matrix, which is a fibrous protein network extending throughoutthe nuclear interior. A protamine gene cluster is located within 30 kbof the SOCS-1 gene. The matrix association at SOCS-1 and PRM1 arealtered between sperm cells and somatic cells (Kramer et al., supra,1998), suggesting that the matrix association of SOCS-1 is alteredbetween the SOCS-1 methylated and unmethylated cells.

The matrix association at 3 loci in SOCS-1 methylated cells and SOCS-1unmethylated cells was examined. The Actin B (ACTB) locus was used as acontrol for DNA attached to the nuclear matrix. To analyze the SOCS-1and PRM1 loci, primers were designed to examine the 5′ regions of thesegenes, where the functional matrix association was demonstrated (Krameret al., supra, 1998). The two primer sets are separated by 38 kb. At theACTB locus, a pellet fraction composed of matrix associated DNA showedequal intensity between the SOCS-1 methylated and unmethylated cells. Atthe PRM1 locus, an equal amount of matrix association also was observedin SOCS-1 methylated and unmethylated cells. At the SOCS-1 locus, incontrast, matrix associated DNA from the SOCS-1 unmethylated cells wasgreater than that from the SOCS-1 methylated cells (see FIG. 2 inYoshikawa et al., supra, 2001). These results indicate that matrixassociation of the SOCS-1 gene was reduced in the SOCS-1 methylatedcells.

Analysis of SOCS-1 CpG Island Methylation in Primary HCC Tissue Samples

Methylation of the SOCS-1 CpG island was examined in 26 surgicallyresected primary HCC samples. Of the 26 samples, five were paired(tumor/non-tumor); only tumor tissue was available for the remainingsamples. None of the non-tumor liver samples showed methylation ofSOCS-1, whereas methylation of SOCS-1 was detected in 4 of the 5 tumorsamples from these paired samples. Methylation also was detected in 13of the 21 samples for only tumor tissue was available (see FIG. 3 inYoshikawa et al., supra, 2001). Overall, 17 of 26 (65%) of the primaryHCC samples were methylated in the SOCS-1 CpG island. These resultsdemonstrate that the methylation status of the SOCS-1 gene can be usedto distinguish normal liver from hepatocellular carcinoma.

Constitutive JAK2 Activation in HCC Cells Containing MethylationSilenced SOCS-1

The functional consequence of SOCS-1 methylation-related silencing alsowas examined. Examination of the tyrosine phosphorylation status of JAK2in HCC cell lines revealed that JAK2 was not phosphorylated in a cellline that was free of methylation in the CpG island and expressedSOCS-1, whereas two SOCS-1 methylation-silenced cell lines showed JAK2phosphorylation (see FIG. 4a in Yoshikawa et al., supra, 2001). Theseresults are consistent with JAK2 inhibition by SOCS-1, and withconstitutive activation of JAK2 in cells exhibiting inactivation ofSOCS-1 gene expression due to CpG island methylation.

To further determine the role of SOCS-1 in the JAK/STAT pathway, thephosphorylation status of STAT3 was examined. It was expected that JAK2activation would result in constitutive STAT3 phosphorylation and,indeed, three cell lines (SNU-387, HuH-1, and PLC/PRF/5) that exhibitedSOCS-1 inactivation and JAK2 phosphorylation also showed constitutivephosphorylation of STAT3 (see FIG. 4b in Yoshikawa et al., supra, 2001).In comparison, in two cell lines without SOCS-1 inactivation, one(HuH-7) had no constitutive phosphorylation of STAT3 and the other(SNU-182) showed STAT3 phosphorylation. This result indicates that, atleast in the SNU-182 cell line, STAT3 is activated by pathways otherthan JAK2, which was not phosphorylated.

To confirm these results, cell lines were treated with the JAK2 specificinhibitor, AG490 (Meydan et al., Nature 379:645-648, 1996, which isincorporated herein by reference). AG490 treatment led to a timedependent reduction in STAT3 phosphorylation in SOCS-1 inactivated (JAK2activated) cell lines (SNU-387 and HuH-1), but not in the SOCS-1expressing SNU-182 cells. These results demonstrate that SOCS-1inactivation is associated with activation of the JAK/STAT pathway,including the well characterized STAT3 protein.

Growth Suppression by SOCS-1 Restoration

Because the JAK/STAT pathway may be an oncogenic pathway (Ihle et al.,supra, 1995), the role of SOCS-1 in cell growth was examined. When aSOCS-1 expression vector was introduced into HCC cells exhibitingmethylation-silenced SOCS-1 gene expression, the number of colonies ofSOCS-1 transfected cells was substantially decreased as compared withHCC cells transfected with the control vector (see FIG. 5 in Yoshikawaet al., supra, 2001).

AG490 has been reported to suppress growth of B cell leukemia cells, inwhich JAK2 was constitutively activated (Meydan et al., supra, 1996). Totest for the growth suppression by AG490, HCC cells exhibitingmethylation silenced SOCS-1 gene expression were grown in the presenceor absence of AG490. Incubation with AG490 effectively suppressed thegrowth of the SOCS-1 methylation-silenced cells as shown by a decreasednumber of colonies. This result confirms the constitutive activation ofJAK2 in the SOCS-1 methylation-silenced HCC cell lines, and demonstratesthat constitutive activation of the JAK/STAT pathway is associated withsilencing of SOCS-1 by methylation in HCC.

To examine the mechanism of growth suppression by SOCS-1, SOCS-1methylation-silenced cells were transiently transfected with the SOCS-1expression vector, and the expression of SOCS-1, the presence offragmented DNA, and the integrity of cell nuclei were examined using anantibody to a tag on the expressed SOCS-1 protein, the TUNEL assay, andDAPI staining, respectively. SOCS-1 expressing cells selectivelydemonstrated fragmented genomic DNA, whereas cells that were nottransfected with SOCS-1 had intact nuclei (see FIG. 5 in Yoshikawa etal., supra, 2001). These results indicate that restoration of SOCS-1selectively induces apoptosis in SOCS-1 methylation-silenced cells.

The effect of SOCS-1 restoration in HCC cells also was examined forgrowth in soft agar. A SOCS-1 methylation-silenced cell line transfectedwith the SOCS-1 expression vector, and incubated in soft agar added withselection medium for 4 weeks, showed decreased colony numbers comparedwith the control transfectants. These results indicate that SOCS-1suppresses anchorage-independent growth and growth in monolayers, andthat restoration of SOCS-1 expression suppresses cell growth by inducingapoptosis in HCC cells.

Aberrant methylation in the SOCS-1 CpG island was identified in 65% ofprimary HCC samples and was tightly linked to the silencing of geneexpression. In addition, an alteration of the matrix attachment of theSOCS-1 locus was detected, thus associating the methylation andexpression status and indicating that DNA methylation facilitatesdynamic changes in chromatin structure. The high incidence ofmethylation-associated SOCS-1 gene inactivation in HCC indicates thatSOCS-1 silencing is involved in the etiology of HCC.

Activation of the JAK/STAT pathway has been implicated in the promotionof cancer. For example, activation of Drosophila JAK causedhematopoietic neoplasia, and a self-activating STAT3 mutation causedcellular transformation and tumorigenicity in nude mice. In addition,constitutive activation of the JAK/STAT pathway was reported intransformed cells and cancer cells, and JAK2 was constitutivelyactivated in B cell leukemia. In T cell lines, human T celllymphotrophic virus (HTLV-1) infection constitutively activated JAK1,JAK3, STAT3 and STAT5, and the v-abl oncogene constitutively activatedJAK1 and JAK3 in transformed murine pre-B cells; the v-abl protein wasphysically associated with JAK1 and JAK3 in these pre-B cells (Danial etal., Science 269:1875-1877, 1995). STAT3 constitutive activation alsowas reported in v-src, v-fps, and v-sis transformed mouse fibroblastcell lines, and in human breast carcinoma cell lines (Garcia et al.,supra, 1997).

A differential pattern of STAT activation was observed among lymphoid,myeloid and lymphoma cells. STAT5 was predominantly activated in acutelymphoid leukemia, whereas STAT1 and STAT3 were activated in acutemyeloid leukemia and Burkitt's lymphoma (Weber-Nordt et al., Blood88:809-816, 1996). A role for the JAK/STAT pathway in oncogenesis alsowas suggested by liver regeneration experiments, in that STAT3 DNAbinding activity was greatly increased in the remnant rat liver afterpartial hepatectomy, and STAT3 translocated into the nucleus (Cressmanet al., Hepatology 21:1443-1449, 1995; Trautwein et al.,Gastroenterology 110:1854-1862, 1996). Additional support for the roleof SOCS-1 in hepatocytes comes from reports of SOCS-1 knock out mice,which die of liver degeneration and lymphoid deficiency between 2 and 3weeks after birth (Starr et al., Proc. Natl. Acad. Sci. USA95:14395-14399, 1998; Naka et al., Proc. Natl. Acad. Sci. USA95:15577-15582, 1998).

As disclosed herein, SOCS-1 methylation silencing was associated withJAK2 constitutive activation in HCC cell lines, thus providing a meansby which aberrant regulation of the JAK/STAT pathway is involved inoncogenesis. Stimulation of the JAK/STAT pathway by cytokines and growthfactors results in transactivation of target genes. In normal cells,SOCS-1 is simultaneously activated and blocks JAK activation, leading totermination or attenuation of the signal. The results disclosed hereinindicate that in cancer cells, SOCS-1 is silenced by methylation and,therefore, is unable to terminate the signal, resulting in constitutiveJAK activation. As a result, SOCS-1 silenced cells can exhibit unopposedgrowth stimulation due, for example, to the action of cytokines, growthfactors and hormones that stimulate the JAK/STAT pathway.

The present results indicate that SOCS-1 normally functions to suppressgrowth of hepatocytes, as revealed in the decreased growth of SOCS-1restored HCC cells in monolayer and in soft agar culture. Further,apoptosis occurred in the SOCS-1 restored cells, thus accounting, atleast in part, for the growth suppression. The growth suppressionactivity of SOCS-1 in the JAK/STAT pathway was further supported by theobservation that AG490, a specific chemical JAK2 inhibitor, replacedSOCS-1 function. AG490 suppressed the growth of SOCS-1 silenced HCCcells, and reversed the constitutive phosphorylation of STAT3. Notably,AG490 does not suppress the growth of normal haematopoietic progenitorcells at concentrations similar to those used in the present studies(Meydan et al., supra, 1996), and, as disclosed herein, had no effect onnormal liver cells.

A high incidence of the methylation of SOCS-1 and its functionalsignificance indicate that SOCS-1 inactivation can be involved in othercancer, particularly those for which JAK/STAT is constitutivelyactivated (see, for example, Garcia et al., supra, 1997; Meydan et al.,supra, 1996; Weber-Nordt et al., supra, 1996). SOCS-1 also is a memberof the CIS family, which includes several other members, includingSOCS-2, SOCS-3 and CIS2, all of which are negative regulators ofcytokine signal transduction, and the inactivation of which can beinvolved in the development of cancer similar to SOCS-1.

EXAMPLE 2 Methylation of SOCS-1 is Associated with Multiple Myeloma

This Example demonstrates that SOCS-1 gene methylation silencing alsooccurs in multiple myeloma (MM) cells.

Methods

Multiple Myeloma Patient and Control Samples

After informed consent was given, bone marrow (BM) specimens wereaspirated during routine clinical assessment of 35 MM patients, whopresented at the University Hospital Aachen, Germany between 1995 and2000. MM diagnosis and staging classification were made in accordancewith standard criteria (Durie, Sem. Oncol. 13:300-309, 1986). Twocontrol bone marrow aspirates were obtained from patients withnon-metastatic solid tumors or malignant lymphoma without bone marrowinfiltration or hematopoietic dysfunction as part of the routine stagingprocedure. Peripheral blood (PB) samples were collected from fivehealthy volunteers. Mononuclear cells from BM and PB were separated bydensity gradient centrifugation prior to further analysis. Details ofthe lymphoma samples have been described (Katzenellenbogen et al.,supra, 1999; Esteller et al., J. Natl. Canc. Inst. 94:26-32, 2002, eachof which is incorporated herein by reference).

Cell Culture and Drug Treatment

HL60, U266 and Raji cell lines were obtained from the American TypeCulture Collection. KG1a and K562 cell lines were obtained from theGerman Collection of Microorganisms and Cell Cultures. KG1a and HL60cells were cultured in Iscoves's modified Dulbecco's medium (IMDM,Invitrogen Corp.) with 20% FCS (Gemini Bio-Products). U266 cells werecultured in RPMI 1640 (Invitrogen Corp.) with 15% FCS. K562 and Rajicells were cultured in RPMI 1640 with 10% FCS, and XG1 cells werecultured in IMDM with 20% FCS and 10 U/ml IL-6 (Roche).

For gene expression studies, U266 and XG1 cells were incubated incomplete culture medium with a final concentration of 1.0 μM5-azacytidine (Sigma). After 4 days, drug-treated and untreated cellswere harvested and subjected to RNA extraction. In order to assess thesensitivity to AG490, cell lines K562, U266 and XG1 were incubated withor without a final concentration of 50 μM AG490 for 4 days prior toanalysis. For STAT3 phosphorylation analysis, cell lines U266 and XG1were initially starved for 16 hr in culture medium supplemented withonly 1% FCS and no cytokine. Cells were then washed in cold PBS andresuspended in their appropriate complete medium with or without 50 μMAG490 and samples were harvested for protein analysis after 1 hr and 24hr.

Sodium Bisulfite Treatment and Methylation-Specific Polymerase ChainReaction

For SOCS-1 MSP analysis, approximately 1 μg of DNA was modified bytreatment with sodium bisulfite and amplified using primers as describedin Example 1. Normal DNA from PB was treated in vitro with SSS Imethyltransferase (New England Biolabs) in order to generate a positivecontrol for methylated alleles of SOCS-1 (Esteller et al. Cancer Res.59:793-797, 1999, which is incorporated herein by reference).

RNA Isolation and Reverse Transcriptase Polymerase Chain Reaction(RT-PCR)

Total RNA was isolated using a commercially available kit (Qiagen)according to the manufacturer's instructions. Approximately 3 μg RNA persample were reverse transcribed with SuperScript™ reverse transcriptase(Invitrogen). For PCR, 1 μl of the cDNA preparation was amplified usingthe following SOCS-1 primers:

5′-CCCGGAGCATGCGCGAGAGC-3′, (sense; SEQ ID NO: 16) and (antisense; SEQID NO: 17) 5′-TGCGGGCTCTGCTGCTGTGG-3′;and the following GAPDH primers:

(sense; SEQ ID NO: 18) 5′-GACCACAGTCCATGCCATCAC-3′, and (antisense; SEQID NO: 19) 5′-GTCCACCACCCTGTTGCTGTA-3′.

Reactions were hot-started at 95° C. for 5 min and held at 80° C. beforeaddition of 1.25 U of Taq polymerase (Invitrogen Corp.). Temperatureconditions for SOCS-1 were 26 cycles of 95° C., 30 sec; 64° C., 30 sec;and 72° C., 30 sec, followed by 1 cycle of 72° C. for 5 min; and forGAPDH 23 cycles of 95° C., 1 min; 63° C., 1 min; and 72° C., 1 min;followed by 1 cycle of 72° C. for 5 min.

Flow Cytometric Analysis of Apoptosis

Apoptosis was assessed by annexin V binding and counterstaining withpropidium iodide (PI) using a commercially available kit (Pharmingen)according to the manufacturer's instructions. Fluorescence analysis wasperformed on a BD LSR flow cytometer (Becton Dickinson).

Western Blot Analysis

Cell lysis and western blot analysis for STAT3 phosphorylation wereperformed as described in Example 1 (see, also, Yoshikawa et al., supra,2001).

Results

SOCS-1 Methylation Status in Hematopoietic Cell Lines and Normal Tissues

SOCS-1 methylation status was analyzed by MSP in various humanhematopoietic cell lines. SOCS-1 hypermethylation was observed in bothIL-6-dependent MM cell lines, U266 and XG1, and in the AML cell line,KG1a (see, also, Example 3). In comparison, no aberrant methylation wasobserved in the HL60 AML cell line, or in the K562 chronic myelogenousleukemia (CML) cell line, or the Burkitt's lymphoma cell line (Raji);and no SOCS-1 methylation was found in normal peripheral bloodmononuclear cells (PBMNC; n=5) or in non-malignant bone marrow cells(n=2).

SOCS-1 Expression in MM Cell Lines

The MM cell lines were examined by semi-quantitative RT-PCR. XG1 cellsrequire the addition of exogenous IL-6 (Zhang et al., Blood83:3654-3663, 1994), whereas U266 cells depend on an IL-6 autocrine loop(Schwab et al., Blood 77:587-593, 1991). Despite the presence of IL-6 inthe cell culture medium, the MM cell lines showed low (U266) orundetectable (XG1) SOCS-1 transcript levels. In comparison, normal PBMNCincubated in IMDM with 20% FCS and 10 U/ml IL-6 for 2 hr showed strongSOCS-1 expression; unstimulated PBMNC expressed only low levels ofSOCS-1. Incubation of U266 and XG1 cells with 1 μM 5-azacytidine for 96hr resulted in reactivation of SOCS-1 gene expression in both MM celllines, confirming that methylation was responsible for the loss ofSOCS-1 expression in the MM cells.

Sensitivity of Hematopoietic Cell Lines to AG490

In order to investigate the activation of the JAK/STAT pathway in thecontext of methylation-silencing of SOCS-1 in hematopoietic cell lines,K562, U266 and XG1 cells were treated with 50 μM AG490 and thepercentage of apoptotic cells after 96 hr was detected by annexin Vbinding and PI counterstaining. AG490 treatment induced apoptosis inU266 and XG1 cells, but had only a small effect on K562 cells. Theseresults indicate that cell lines that carry a hypermethylated SOCS-1gene, including MM cells and HCC cells (see Example 1) have a greatersensitivity to apoptosis induction by AG490 as compared to cell lineswithout SOCS-1 hypermethylation.

STAT3 Phosphorylation

AG490 treatment also inhibited STAT3 phosphorylation in U266 cells,while there were only minor effects in XG1 cells. These resultsdemonstrate that, in addition to inducing apoptosis in the MM celllines, AG490 treatment also results in inhibition of STAT3phosphorylation, which represents a downstream event in the JAK/STATpathway.

SOCS-1 Methylation in Primary Patient Samples

SOCS-1 methylation status also was examined by MSP in primary patientsamples. Aberrant methylation was present in 62.9% (23/35) of MM cases.Since IL-6 is involved in B cell growth and differentiation into plasmacells, and acts as a survival factor in MM pathogenesis, an associationof methylation-silencing of SOCS-1 with other lymphoid malignancies wasexamined. MSP analysis of malignant lymphomas of various histologies,including 55 B cell lymphomas, 4 T cell lymphomas, and 3 Hodgkin'slymphomas revealed SOCS-1 hypermethylation in only 3.2% (2/62) of thesamples, including one large B cell lymphoma and one T lymphoblasticlymphoma.

Stimulation of the JAK/STAT pathway by cytokines or growth factors suchas IL-6 results in transactivation of target genes. Under physiologicalconditions, SOCS-1 is simultaneously up-regulated and blocks JAKactivation, leading to termination or attenuation of the signal. Ifhypermethylation-associated transcriptional silencing of SOCS-1 occursin tumor cells, this negative feedback loop is disrupted, and the cellscan become more responsive to stimulation by cytokines and growthfactors that use the JAK/STAT pathway for signal transduction. In MMpathogenesis, IL-6 has been identified as an essential growth andsurvival factor. As such, loss of SOCS-1 function can result inincreased IL-6 signal transduction, and support survival and expansionof MM cells.

As disclosed herein, the IL-6-dependent MM cell lines U266 and XG1exhibited aberrant SOCS-1 hypemethylation that is associated withtranscriptional silencing, and treatment of these cell lines with thedemethylating agent, 5-azacytidine, resulted in re-expression of SOCS-1.The importance of JAK activation in cell lines containing ahypermethylated SOCS-1 gene was demonstrated by their sensitivity to thechemical JAK inhibitor, AG490. These results are in accordance with thefinding that, in HCC cell lines, silencing of SOCS-1 by hypermethylationwas associated with constitutive activation of JAK2 (Example 1). AG490also resulted in inhibition of STAT3 phosphorylation in U266 cells,similar to the effect described in the MM cell line, XG2 (see De Vos etal., Brit. J. Haematol. 109:823-828, 2000, which is incorporated hereinby reference).

Aberrant methylation within the SOCS-1 CpG island was identified in62.9% of MM patient samples, but in only 3.2% of primary tissues fromdifferent malignant lymphomas. This finding is in accordance with theimportance of the JAK/STAT pathway, particularly IL-6 signaling, in MMpathogenesis compared to other lymphoid neoplasms. Previous molecularstudies have focused primarily on genetic aberrations in MM, whereinchanges that were detected included chromosomal translocations involvingthe immunoglobulin heavy chain locus on chromosome 14q32 and variouspartner genes such as cyclin D1, cyclin D3, fibroblast growth factorreceptor 3 and c-maf, as well as mutations of N-ras and K-ras (seeHallek et al., Blood 91:3-21, 1998, Kastrinakis et al., Ann. Oncol. 11:1217-1228, 2000). Hypermethylation of p15^(INK4B), p16^(INK4A), andDAP-kinase also has been identified in MM patients and cell lines (Ng etal., supra, 2001; Ng et al., Blood 89:2500-2506, 1997; Urashima et al.,Clin. Cancer Res. 3:2173-2179, 1997; Tasaka et al., Brit. J. Haematol.101:558-564, 1998; Guillerm et al., Blood 98:244-246, 2001),demonstrating that epigenetic silencing of genes that effect cell cycleregulation and apoptosis pathways is an additional mechanism of geneinactivation in MM.

The results disclosed herein reveal the presence of an importantepigenetic event in the pathogenesis of MM. Methylation-silencing ofSOCS-1 gene expression can result in increased responsiveness of theneoplastic clone to IL-6 signaling, thus supporting survival andexpansion of MM cells. As such, the present results not only provide aninsight into the pathogenesis of MM, but, becausehypermethylation-associated gene silencing is a reversible phenomenon,also provide indications for treating MM (Cameron et al., Nat. Genet.21:103-107, 1999). For example, demethylating agents such as5-azacytidine, which are clinically effective in patients with AML andmyelodysplastic syndrome (see, for example, Willemze et al., Leukemia 11(Suppl. 1):S24-27, 1997, describing an EORTC Leukemia Cooperative Groupphase II study (06893); Wijermans et al., J. Clin. Oncol. 18:956-962,2000, describing a multicenter phase II study in elderly patients, eachof which is incorporated herein by reference), are now indicated fortreatment of MM patients having methylation-silenced SOCS-1 geneexpression.

The high prevalence of SOCS-1 hypermethylation in MM indicates that thispathway can be targeted for therapeutic intervention. While reactivationof genes like p15^(INK4B) and p16^(INK4A) can contribute toreconstitution of disrupted cell cycle regulation, reactivation ofSOCS-1 can functionally restore the endogenous negative feedback loop ofIL-6 signaling. Since IL-6 contributes to MM resistance to conventionaltherapy options such as corticosteroids, inhibition of IL-6 signaling bySOCS-1 reactivation can be used to overcome such resistance.

The recognition that IL-6 is a survival factor for MM cells has promptedthe development of strategies to block its effects therapeutically, forexample, using anti-IL-6 monoclonal antibodies or antisenseoligonucleotides. As an alternative approach, in MM cases in whichSOCS-1 is silenced by hypermethylation, treatment with demethylatingagents can be used to reconstitute the function of SOCS-1 as aphysiological suppressor of IL-6 signaling. Thus, the combination ofdemethylating agents with conventional therapeutics or tyrosine kinaseinhibitors can provide a promising new strategy in MM therapy.

EXAMPLE 3 Methylation of SOCS-1 is Associated with Leukemia

This Example demonstrate that hypermethylation of the SOCS-1 gene alsois associated with acute leukemias.

Methods

Primary Leukemia Samples

Adult leukemia were obtained from the University Hospital Aachen,Germany. The pediatric have previously been described for inactivationat the INK4A and INK4B locus (Herman et al., supra, 1997).

Cell Culture and Drug Treatment

HL60 cells were obtained from the American Type Culture Collection andKG1a cells from the German Collection of Microorganisms and CellCultures. Both cell lines were routinely cultured in IMDM (InvitrogenCorp.) with 20% FCS (Gemini Bio-Products). In order to assess thesensitivity to AG490, HL60 and KG1a cells were incubated with or withouta final concentration of 50 μM AG490 (Sigma) for 96 hr prior toanalysis.

Flow Cytometric Analysis of Apoptosis

The percentage of apoptotic cells was determined by annexin V-bindingand counterstaining with propidium iodide (PI), and fluorescenceanalysis was performed as described in Example 2.

Methylation Specific PCR

MSP was performed as described in Example 1. The methylation specificprimer sequences were as follows:

(forward; SEQ ID NO: 2) 5′-TTCGCGTGTATTTTTAGGTCGGTC-3′, and (reverse;SEQ ID NO: 3) 5′-CGACACAACTCCTACAACGACCG-3′.The unmethylation specific primer sequences were as follows:

(forward; SEQ ID NO: 4) 5′-TTATGAGTATTTGTGTGTATTTTTAGGTTGGTT-3′, and(reverse; SEQ ID NO: 5) 5′-CACTAACAACACAACTCCTACAACAACCA-3′.Western Blot Analysis

Western blot analysis was performed as described in Example 1. Blotswere stained using anti-phospho-STAT1, anti-phospho-STAT3 oranti-phospho-STAT5 antibody (New England Biolabs) to detect tyrosinephosphorylation of STAT protein. After removing the antibody, the blotwas analyzed using anti-STAT1, anti-STAT3 or anti-STAT5 antibody,respectively (BD Transduction Laboratories).

Results

Methylation of SOCS-1 CpG Island in Acute Leukemia.

SOCS-1 CpG island methylation was examined in 38 pediatric leukemia and51 adult leukemia samples. The 38 pediatric leukemia included 17 acutemyeloid leukemia (AML), 8 T cell ALL (T-ALL) and 13 B cell ALL (B-ALL).The 51 adult leukemia included 37 de novo AML and 14 AML transformedfrom myelodysplastic syndrome (MDS). The overall frequency of SOCS-1methylation was 31% (28 of 89). In pediatric leukemia, methylation wasdetected in 6 of 17 (35%) AML, 2 of 8 (25%) T-ALL and 2 of 13 (15%)B-ALL; and for adult leukemia, methylation was detected in 11 of 37(30%) de novo AML and 7 of 14 (50%) secondary AML transformed from MDS.No methylation was detected in 2 MDS samples without transformation.Methylation of SOCS-1 was found in all subtypes of AML (M0-M7).

Effect of JAK/STAT Inhibition in Leukemia

The effect of SOCS-1 methylation on inhibition of JAK/STAT pathway inleukemia was examined. Treatment of the AML cell line, KG1a, which wasmethylated in the SOCS-1 CpG island, with AG490 induced apoptosis,whereas treatment of the AML line, HL60, in which SOCS-1 was notmethylated, had no effect, as determined by annexin V binding and PIcounterstaining. These results demonstrate that the leukemia cell linewith a hypermethylated SOCS-1 gene showed greater sensitivity to AG490as compared to cells without SOCS-1 hypermethylation.

The effect of AG490 on STAT activation in the SOCS-1 methylated cellsalso was examined following treatment with AG490. Constitutiveactivation of STAT1, STAT3 and STAT5 was detected in the cell line withSOCS-1 methylation prior to treatment with AG490, whereas, followingtreatment with AG490, tyrosine phosphorylation of STAT3 and,particularly, of STAT1 decreased; no change was observed in the tyrosinephosphorylation of STAT5.

These results demonstrate that hypermethylation of the SOCS-1 gene alsois associated with acute leukemia, and that, in addition to geneticalterations, activation of the JAK/STAT pathway in leukemias isassociated with an epigenetic change. Hypermethylation-associatedsilencing of SOCS-1 can increase activation of the JAK/STAT signaltransduction pathway, thus supporting the growth of leukemia cellclones. The importance of the JAK/STAT pathway in KG1a cells, whichcontain a hypermethylated SOCS-1 gene, is underscored by theirsensitivity to AG490, which inhibits JAK activity. In contrast, AG490showed only weak induction of apoptosis in HL60 cells, which have anunmethylated SOCS-1 gene but contain an activating N-ras mutation.Previous reports have demonstrated the effectiveness of AG490 in thetreatment of ALL (Meydan et al., Nature 379: 645-648, 1996). Theimportance of SOCS-1 inactivation in hematological malignancies issupported by reports that SOCS-1 suppresses the expansion of immaturethymocytes (Trop et al., Blood 97: 2269-2277, 2001), and that AG490induces apoptosis in myeloma cells (De Vos et al., Brit. J. Haematol.109: 823-828, 2000; Catlett-Falcone et al., supra, 1999) and leukemicSezary cells (Eriksen et al., Leukemia 15: 787-793).

Aberrant methylation of the SOCS-1 CpG island was detected in manydifferent subtypes of acute leukemia, with no marked difference betweenpediatric and adult leukemias. Methylation of SOCS-1 occurred in 31% ofde novo AML, 50% in secondary AML, 25% in T ALL, and 15% in B ALL cases.Thus, SOCS-1 methylation occurs with similar incidence in all 3 types ofde novo acute leukemia, although the secondary AML demonstrated arelatively higher incidence. These results indicate that SOCS-1methylation is ubiquitous among acute leukemia, and that the involvementof the JAK/STAT signal transduction pathway is significant in thedevelopment of hematological malignancies. The secondary AML transformedfrom MDS showed the higher methylation incidence than de novo leukemia,and 2 of 2 MDS without transformation were free from the aberrantmethylation. The distribution of SOCS-1 methylation also was analyzedamong subtypes of AML in adult. One or 2 positive samples appeared inmost of the subtypes both in de novo and secondary AML, furtherindicating that SOCS-1 methylation occurs in various cell types.Together with the similar occurrence among AML, T ALL and B ALL, theseresults indicate that SOCS-1 methylation can be used as a marker for anykind of acute leukemia.

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A method for selecting an agent for treating a cancer patient, themethod comprising detecting decreased expression of SOCS-1 gene productdue to methylation of a cytosine residue of a CpG dinucleotide in a CpGisland of a suppressor of cytokine signaling-1 (SOCS-1) gene in a samplecomprising cancer cells from the patient; and selecting an agent thatrestores the expression of the SOCS-1 gene product in the cancer cells.2. The method of claim 1, wherein the SOCS-1 gene product is encoded bySEQ ID NO:1.
 3. The method of claim 1, wherein the agent comprises ademethylating agent.
 4. The method of claim 3, wherein the demethylatingagent comprises 5-aza-2′-deoxycytidine.